Strings of epitopes useful in diagnosing and eliciting immune responses to sexually transmitted infections

ABSTRACT

Methods and compositions for detecting and diagnosing sexually transmitted infections using a string of epitopes (SOE) specific for detection of causative microorganisms are provided. The antigenic epitopes may be single epitope sequences, a plurality of epitope sequences joined by amino acid linkers to form a series of epitopes (SOE), or nucleotide sequences encoding one or more SOEs and host cells harboring said SOE nucleotide sequences. SOEs specific for highly immunogenic regions of proteins from Trichomonas, Treponema and Neisseria species are provided. SOEs to detect the presence of Trichomonas species comprise regions from Trichomonas aldolase, GAPDH, α-enolase and α-actinin proteins. Pharmaceutical compositions comprising SOEs can also be used as vaccines or to elicit an immune response to specific microorganisms.

FIELD OF THE INVENTION

The invention relates to compositions and methods used to detect and/ordiagnose infections caused by, for example, Trichomonas, Treponema, andNeisseria species. The invention further relates to compositions andmethods for eliciting an immune response and/or vaccinating againstinfection by Trichomonas, Treponema, and Neisseria species.

BACKGROUND OF THE INVENTION

Sexually transmitted infections (STIs) are a major global cause of acuteillness, infertility, long term disability and death, with severemedical and psychological consequences for millions of men, women andinfants. WHO estimated that 340 million new cases of syphilis,gonorrhoea, Chlamydia and trichomoniasis occurred throughout the worldin 1999 in men and women aged 15-49 years, and incidence has risensteadily since then.

Trichomonas vaginalis causes vaginitis in women and non-gonococcal,non-chlamydial urethritis in men. Among men, the most recent findingsindicate a relationship between seropositivity to T. vaginalis andprostate cancer. This parasite is now the number one, non-viral sexuallytransmitted disease agent. In 2013, the incidence of this sexuallytransmitted infection (STI) referred to as trichomonosis ortrichomoniasis is estimated to be 10 million women in the United Statesand 270 to 350 million women worldwide. Health consequences to womeninclude cervical cancer, pelvic inflammatory disease, infertility,increased HPV and herpes susceptibility, and adverse pregnancy outcomesaccompanied with low-birth-weight infants. Significantly, 25% of HIVseroconversions are the direct result of trichomonosis, which is knownto increase the portal of exit and entry of HIV infectious viralparticles. Therefore, control of trichomonosis may be one of the mosteffective means of reducing HIV transmission risk and of preventingprostate and cervical cancers worldwide.

It is clear that the public health costs as a result of this STI areenormous, and interference strategies are needed. The most importantinterference strategy is the availability of rapid, accurate diagnosticswith exceptional sensitivity and specificity toward this STI agent.Despite the impact of this STI to public health, fundamental aspects ofT. vaginalis cell biology and mechanisms of pathogenesis remain unknown.As previously disclosed in U.S. Pat. No. 8,017,103 B2, incorporated byreference herein, α-actinin is expressed by Trichomonas species and canbe used to detect the presence of Trichomonas infection. However, theantibodies to parasite proteins available hitherto are inferior in theirability to detect the immunoreactive trichomonad protein antigens.

SUMMARY OF THE INVENTION

The antibodies, proteins, and epitopes to the proteins detected by thehuman antibody described herein are novel and have increased utility fordiagnostics to STIs. Antigenicity and specificity is increased with themicroorganism-specific target protein antigens and epitopes describedherein compared to previously available diagnostics. Furthermore, thepresent disclosure overcomes prior shortcomings in the art by providingepitopes for detecting antibody in sera of humans exposed to and/orinfected with T. vaginalis and other microorganisms that cause STIs,such as Treponema pallidum and Neisseria gonorrhoeae. In addition tocompositions and methods relating to detection and diagnosis of STIs,embodiments of the disclosure includes compositions and methods foreliciting an immune response and providing vaccines that can protectsubjects from STIs.

An embodiment of the disclosure is a method of detecting the presence ofa microorganism in a biological sample from a subject, comprising thesteps of identifying at least one protein that is expressed by themicroorganism of interest, determining regions of the protein that arehighly immunogenic, and designing epitopes encoding those regions. Insome embodiments, the epitopes are embedded within a 15-mer peptide. Thedisclosure is further directed to synthesizing a plurality of epitopesin a linear array to form a series of epitopes (SOE), wherein theepitopes are joined with amino acid linkers. In some embodiments, thelinkers are a number of any amino acid residues. In some embodiments,the linkers are repeats of amino acid residues such as glycine (-GG-),lysine (-KK-), glutamic acid (-EE-), and mixtures thereof within theSOE. The SOE is then contacted with a biological sample under conditionswhereby an antigen-antibody complex can form, and formation of at leastone said antigen-antibody complex is an indication of the presence ofthe microorganism of interest. The SOE typically comprises at least sixof said epitopes, but may comprise fewer or greater numbers of epitopes.A composition may comprise SOEs to detect multiple proteins from asingle species or family of microorganisms, or from a group of unrelatedmicroorganisms.

Sequences encoding epitopes and SOEs are provided to detect, diagnose ortreat infections caused by Trichomonas, Treponema, and Neisseriaspecies. Aspects of the disclosure are applicable to other species.Exemplary SOEs detect Trichomonas species including Trichomonas (T.)vaginalis, T. vaginalis isolates T016, T068-II, UT40, and VB102,Tritrichomonas (Tt.) foetus, T. foetus, Tt enteris, T. paviovi, Tt.suis, Tt. rotunda, T. buttreyi, Tt. ovis, Tt. equi, T. equibuccalis, T.anatis, Tt. eberthi, T. gallinae, T. gallinarum, Tt. caviae, Tt muris,Tt. wenoni, Tt. Minuta, T. microti, T. canistomae, T. felistomae, T.tenax, Tt. hominis, and T. macacovaginae. Epitopes, 15-mer epitopes andSOE sequences are provided to detect, diagnose, or treat infectionscaused by Treponema pallidum and Neisseria gonorrhoeae.

Additional bacterial pathogens may be detected, diagnosed, or vaccinatedagainst, with SOEs encoding highly immunogenic regions of one or moreproteins expressed by a microorganism or bacterial pathogen of interest.Other microorganisms include, but are not limited to Chlamydiatrachomatis, Saccharomyces cerevisiae, Candida albicans, Streptococcuspyogenes, Streptococcus pneumoniae, and Staphylococcus aureus.

In one embodiment, detection is performed by immunoassay. A preferredimmunoassay is an enzyme-linked immunosorbent assay (ELISA). Thebiological sample can be saliva, urine, blood, serum or plasma, a lunglavage or sputum sample, and the subject may be male or female.Biological samples can also be vaginal fluid or washing, or semen orprostatic fluid.

In some embodiments, the biological sample is cerebrospinal fluid, jointfluid, body cavity fluid, whole cells, cell extracts, tissue, biopsymaterial, aspirates, exudates, pap smear samples, pap smearpreparations, slide preparations, fixed cells, and tissue sections. Thebiological samples can be collected from a subject that may be human,non-human primate, dog, cat, cattle, sheep, swine, horse, bird, mouseand rat.

In one exemplary embodiment, a method of diagnosing a Trichomonasinfection in a subject comprises the steps of identifying at least oneprotein that is expressed by a Trichomonas species, determining one ormore regions of at least one protein from Trichomonas that is/are highlyimmunogenic, designing epitopes encoding said regions of the protein,and synthesizing a plurality of epitopes in a linear array to form aseries of epitopes (SOE) wherein the epitopes are joined with an aminoacid linker. For example, any SOE may contain a mixture of two or moreof -GG-, -KK-, and -EE- repeats. A biological sample from a subject iscontacted with at least one SOE that binds an antibody to aTrichomonas-specific protein selected from the group consisting ofaldolase, GAPDH, α-enolase and α-actinin, under conditions whereby anepitope-antibody complex can form, and detecting formation of at leastone epitope-antibody complex as an indication of Trichomonas infection.The biological sample may be serum, plasma, blood, saliva, semen,cerebrospinal fluid, semen, prostatic fluid, urine, sputum, joint fluid,body cavity fluid, whole cells, cell extracts, tissue, biopsy material,aspirates, exudates, vaginal washings, pap smear samples, pap smearpreparations, slide preparations, fixed cells, or tissue sections. Themethod of detecting the epitope-antibody may be performed using animmunoassay. In one exemplary embodiment, the immunoassay is anenzyme-linked immunosorbent assay (ELISA). The Trichomonas species thatcan be identified include Trichomonas (T.) vaginalis, T. vaginalisisolates T016, T068-II, UT40, and VB102, Tritrichomonas (Tt.) foetus, T.foetus, Tt enteris, T. paviovi, Tt. suis, Tt. rotunda, T. buttreyi, Tt.ovis, Tt. equi, T. equibuccalis, T. anatis, Tt. eberthi, T. gallinae, T.gallinarum, Tt. caviae, Tt muris, Tt. wenoni, Tt. minuta, T. microti, T.canistomae, T. felistomae, T. tenax, Tt. hominis, and T. macacovaginae.A subject may be any animal that can be infected by trichomonads. Incertain embodiments, the subject is human.

An exemplary embodiment includes a method of diagnosing in a subject asexually transmitted infection (STI) selected from the group consistingof trichomoniasis, gonorrohoeae, and syphilis. This embodiment involvesthe steps of identifying at least one protein that is expressed by themicroorganism of interest, determining regions of at least one proteinthat is highly immunogenic, designing epitopes encoding the highlyimmunogenic regions of the protein, and synthesizing a plurality of saidepitopes in a linear array to form a series of epitopes (SOE) whereinthe 15-mer epitopes are joined with amino acid linkers. Variations onthis method further comprise assaying biological samples from a subjectthat are collected at two or more different time points. The interval oftime may be days, weeks, or months, as deemed appropriate by one ofordinary skill in the art of STI diagnosis. The assay can be animmunoassay, with at least one SOE encoding at least one proteinspecific to one or more microorganisms suspected of causing a STI, underconditions whereby an epitope-antibody or antigen-antibody complex canform; and detecting formation of at least one epitope-antibody complexin the two or more samples. Detection readout at the first time point iscompared with detection readout of the second or later time point andthe comparison is used to determine the status of a STI in said subject.

Embodiments of the invention include a monoclonal antibody thatrecognizes an epitope selected from the group of ALDwsu-1, ALDwsu-2,ALD12A, ALD64A, B44, ENOwsu-2, ENOwsu-3, ENOwsu-4, ENOwsu-6, B43,GAPwsu-2, GAPwsu-3, and HA423 (Tables 1 and 2A).

Embodiments also include an epitope selected from the group consistingof SEQ ID NO:1-53, 66-78, 104-106, 121-126, 139-143 and 162-174; or15-mer epitope selected from the group consisting of SEQ ID NO:79-102,107-119, and 128-133. The invention is further a string of epitopes(SOE), comprising a plurality of epitopes linked by an amino acidlinker, selected from the group consisting of SEQ ID NO:120, 127, 134,145, 146, and 175.

Embodiments further include a nucleic acid encoding at least oneepitope, or at least one 15-mer epitope, or at least one string ofepitopes (SOE), wherein the protein product of the nucleic acid binds toat least one antibody type in a biological sample and at least oneantibody type is reactive with at least one Trichomonas protein selectedfrom the group consisting of aldolase, alpha-enolase, GAPDH, andalpha-actinin.

Embodiments also include a host cell comprising a transgene encoding astring of epitopes (SOE), wherein the SOE comprises a plurality ofepitopes selected from NO:1-53, 66-78, 104-106, 121-126, AND 139-143, ora plurality of 15-mer epitopes selected from the group consisting of SEQID NO:79-102, 107-119, AND 128-133, wherein each SOE binds to at leastone antibody type in a biological sample and the antibody type isreactive with at least one protein from a microorganism of interest. ForTrichomonas, the protein is selected from the group consisting ofaldolase, alpha-enolase, GAPDH, and alpha-actinin.

In addition, embodiments include a kit for diagnosis of a sexuallytransmitted infection (STI) in a subject, comprising at least one stringof epitopes (SOE) able to bind at least one antibody type in abiological sample that is reactive with at least one protein from amicroorganism selected from the group consisting of Trichomonas,Treponema, and Neiserria species. The kit may comprise one or morereagents to perform an immunoassay of antibody-epitope orantibody-antigen complexes that form when the SOE of the kit contacts atleast one antibody type in a biological sample, and may include asuitable vessel for performing said immunoassay, and a package insertdescribing steps required for performing said immunoassay, whereindetection of an antibody epitope or antibody-antigen complexes isdiagnostic for a STI.

Embodiments also include eliciting an immune response to a microorganismin a subject. These involve identifying at least one protein that isexpressed by the microorganism, determining regions of said at least oneprotein that are highly immunogenic, designing epitopes encoding saidregions of said at least one protein, and synthesizing a plurality ofsaid epitopes in a linear array to form a series of epitopes (SOE)wherein said epitopes are joined with amino acid linkers. Apharmaceutical composition preferably includes at least one SOE with asuitable carrier and adjuvant, which is administered to a subject in anamount sufficient to stimulate formation of antibodies to the SOE by theimmune system of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to assist in the understanding ofthe invention, but do not the limit the invention and its uses.

FIG. 1 shows an ELISA assay of trichomonads to detect T. vaginalisaldolase, GAPDH, and α-enolase.

FIG. 2 shows detection of T. vaginalis using different amounts ofindividual MAbs to show specificity of the MAbs.

FIGS. 3A, 3B, and 3C show sequence alignment offructose-1,6-bisphosphate aldolase sequences from T. vaginalis (SEQ IDNO:147), T. pallidum (SEQ ID NO:148), N. gonorrhoeae (SEQ ID NO:149), S.pyogenes (SEQ ID NO:150), S. pneumoniae (SEQ ID NO:151), S. aureus (SEQID NO:152), E. coli (SEQ ID NO:153), C. albicans (SEQ ID NO:154),Saccharomyces cerevisiae (SEQ ID NO:155) and Homo sapiens (SEQ IDNO:176).

FIG. 4A shows hydrophobicity and antigenicity profile of T. vaginalisα-enolase (ENO).

FIG. 4B shows hydrophobicity and antigenicity profile of T. vaginalisfructose-1,6-bisphosphate aldolase (ALD).

FIG. 4C shows hydrophobicity and antigenicity profile of T. vaginalisα-glyceraldehyde-3-phosphate dehydrogenase (GAP).

FIGS. 5A, 5B, 5C, and 5D show representative dot blots of individual15-mer peptide epitopes.

FIG. 6A shows representative duplicate dot blots of combinations of15-mer epitopes.

FIG. 6B shows densitometric scans of reactive dot blots from FIG. 4A.

FIG. 7A shows the 111 amino acid sequence of an rSOE encoding 15-merepitopes of GAP (GAPDH), ENO (enolase) and ALD (aldolase) proteins fromT. vaginalis. The sequence is that of SEQ ID NO:145.

FIG. 7B shows SDS-PAGE and Coomassie-brilliant blue stained gels ofrecombinant E. coli, rSOE lysate, flow-through, washes and elutionfractions.

FIG. 8A shows immunodetection of rSOE by ELISA.

FIG. 8B shows immunodetection of rSOE by dot blot.

FIG. 8C shows immunodetection of rSOE by immunoblotting after SDS-PAGE.

FIG. 9 is an example of an SOE comprising thirteen epitopes detected bywomen and men exposed to T. vaginalis are arranged sequentially withinindividual 15-mer peptides separated by a diglycine. The sequence isthat of SEQ ID NO:146.

FIG. 10 shows immunoblot detection of ACT-P2 using IgG1 MAb HA423 andpositive control sera from women and men.

FIGS. 11A and 11B are gels showing that pooled positive control serafrom both women and men are unreactive with HuACTNa and have antibodiesto numerous trichomonad proteins.

FIGS. 12A-12G show SPOTs analysis with positive control sera from womenand men, detecting overlapping peptides on SPOTs membranes of arepresentative epitope and reactions with immobilized ACT-P2 andsynthetic 15-mer peptides used in combination or singly. 12A shows IgGantibody detection of overlapping peptides from spots 214-217. 12B showsthe corresponding amino acid sequences of the individual oligopeptides(SEQ ID NO:162-165). 12C and 12D show signal intensities obtained for 1microgram of ACT-P2 or 2/13M5+W10/M2 immobilized on nitrocellulosemembranes and detected by IgG of positive control sera. 12E showsdensitometric scans of the dots shown in C2 to provide relativeintensities. 12F shows relative reaction of 1 μgram of 15-mer epitopesfrom Table 1, immobilized on nitrocellulose membranes. 12G showsdenitometric scans to provide relative intensities of dots.

FIG. 13 shows a hydrophobicity plot and antigenicity profile for T.vaginalis α-actinin.

FIGS. 14A, 14B, 14C, and 14D show an amino acid sequence alignment of T.vaginalis α-actinin (SEQ ID NO:156) with representative pathogenicorganisms T. suis (SEQ ID NO:177), C. albicans (SEQ ID NO:178), S.cerevisiae (SEQ ID NO:179) and HuACTN1 (SEQ ID NO:157).

FIG. 15 shows an SOE chimeric construct having 18 epitopes unique to T.vaginalis (SEQ ID NO:175).

FIG. 16 shows a coomassie stained gel after SDS-PAGE of Ni-NTA-purifiedAEG:SOE2.

FIG. 17 shows an immunoblot of purified AEG:SOE2 on nitrocelluloseprobed with women/men sera and mouse anti-Tv serum.

FIG. 18 shows a classic 2×2 table for classifying individuals aftertesting.

FIG. 19 shows results of an ELISA comparing negative and positivewomen/men sera and mouse anti-Tv serum for detection of IgG antibody toAEG:SOE2 and ACT-P2 as targets.

DETAILED DESCRIPTION

The present disclosure provides peptide and nucleotide sequencesencoding peptides that are highly immunogenic and may be used to detectthe presence of microorganisms in a biological sample, or diagnose aninfection of the microorganisms in a subject. The methods can comprisedetection of one or more microorganism-specific proteins in one or morebiological samples, or detection of antibodies that a subject hasproduced in response to exposure or infection caused by microorganisms.The disclosure further provides compositions and methods for elicitingan immune response in a subject, or vaccinating against a STI, such astrichomoniasis, gonorrohea or syphilis.

The disclosure further provides methods of arraying the highlyimmunogenic peptides in a linear macromolecule described herein as aseries of epitopes (SOE), recombinant series of epitopes (rSOE), orstring of pearls (SOP). In one embodiment, an SOE is synthetic DNAencoding a protein comprising sequential peptides. The DNA encoding theSOE can be ligated into a plasmid and used to transform or transfect asuitable host cell that will express the SOE as a recombinant protein.In another embodiment, an SOE can be synthesized as a polypeptidesequence encoding sequential peptides, such as 15-mer peptides. Aminoacid linkers, such as repeats of glycine (-GG-), lysine (-KK-), orglutamic acid (-EE-) may be placed between the peptides, either encodedas nucleotides in DNA, or as amino acid residues in a syntheticpolypeptide. In some embodiments, at least six epitopes linked withamino acid linkers are used, however, more epitopes may be added to asynthetic DNA construct or to a synthetic peptide. In anotherembodiment, several SOE species may be included in a composition. Eachof the SOE species may differ in the identity of the epitopes includedin each, or they may further be epitopes from different proteins or evendifferent microorganisms.

The disclosure is based on the unexpected discovery that infection withT. vaginalis and other Trichomonas species can be diagnosed by detectingindividually or in combination T. vaginalis or other Trichomonas speciesaldolase, GAPDH, α-enolase and/or α-actinin proteins or epitopes of theproteins either singly or in combination and/or antibodies to AGEAproteins or epitopes of the proteins either singly or in combination.Similar to embodiments relating to T. vaginalis, further embodiments ofthe disclosure are methods of detecting, diagnosing, treating, orpreventing infection of N. gonorrohoeae and/or T. pallidum. Theseembodiments make use of highly immunogenic peptides, e.g. 15-merpeptides, and SOEs that elicit an immune response to either N.gonorrohoeae or T. pallidum, and sequence listings are provided for eachof the microorganisms of interest. Each of the embodiments of thedisclosure may be practiced by substituting the SOE or antibodiesspecific to a microorganism of interest, such as a species of theTrichomonas, Neiserria or Treponema families. Further, amino acidsequences of other sexually transmitted bacterial pathogens (Chlamydiatrachomatis), of yeast (Saccharomyces cerevisiae and Candida albicans),and of other human bacterial pathogens (Streptococcus pyogenes,Streptococcus pneumoniae, and Staphylococcus aureus) may be identifiedand incorporated into SOEs for detection and diagnosis, and may also beused to elicit immune response and provide protection from infection.

Thus, in some embodiments, the present disclosure provides a method ofdiagnosing a T. vaginalis infection in a subject. Highly immunogenicregions of microorganism-specific proteins selected from the groupconsisting of T. vaginalis aldolase, GAPDH, α-enolase and/or α-actininare identified and at least one SOE comprising peptides encoding thehighly immunogenic regions. In some embodiments, the peptides are 15-merpeptides. In some embodiments, the peptides are linked with an aminoacid linker such as with -GG-, -KK-, or -EE- amino acids. A biologicalsample from the subject suspected of having an infection caused by theT. vaginalis under conditions whereby an antigen/antibody complex canform; and b) detecting formation of an antigen/antibody complex, therebydetecting T. vaginalis AGEA proteins or epitopes of the proteins eithersingly or in combination in the sample and thereby diagnosing a T.vaginalis infection in the subject.

Additionally provided is a method of identifying an acute T. vaginalisinfection in a subject, comprising: a) at a first time point, contactinga first sample from the subject with a T. vaginalis protein selectedfrom a) aldolase, GAPDH, α-enolase and/or α-actinin proteins or epitopesof proteins either singly or in combination, under conditions whereby anantigen/antibody complex can form; b) detecting the formation of anantigen/antibody complex in step a); c) at a second time point,contacting a second sample from the subject with a T. vaginalis proteinor epitopes of proteins selected from aldolase, GAPDH, α-enolase and/orα-actinin proteins, under conditions whereby an antigen/antibody complexcan form; d) detecting the formation of an antigen/antibody complex instep (c); and e) comparing the amount of antigen/antibody complex ofstep (b) with the amount of antigen/antibody complex of step (d),whereby a difference in the amount of antigen/antibody complexidentifies an acute T. vaginalis infection in the subject.

Typically, the biological samples used in practicing the invention arevaginal washings, pap smear or other cell preparations, urine, blood orserum, or saliva samples. However, the sample in all the above variousembodiments of the invention can be any biological fluid or tissue thatcan be used in an immunoassay that either detects antibody in thebiological fluid or detects protein in the biological fluid withavailable polyclonal and/or monoclonal antibodies to the proteins ofthis disclosure, including but not limited to, lung aspirates, semen,cerebrospinal fluid, semen, prostatic fluid, sputum, joint fluid, bodycavity fluid, whole cells, cell extracts, tissue, biopsy material,aspirates, exudates, vaginal washings, pap smear samples, pap smearpreparations, slide preparations, fixed cells, or tissue sections from asubject, where the subject can be either female or male. Several recentreports examining infections in the lungs of immunocompromisedindividuals or patients with acute respiratory distress syndrome haveshown the presence of T. vaginalis as primary or secondary infection.Therefore, it is understood that the invention may be useful fordiagnosis and treatment of patients regardless of STI status, and thatany biological sample may be used.

In the embodiment of identifying an acute infection in a subject, afirst sample is taken at a first time point and a second sample is takenat a second time point and the amount of antibody or antigen and/or thetype of antigen or antibody present in the two samples is compared. Achange in the amount and/or type of antibody or antigen is indicative ofan acute infection and no change in the amount and/or type of antibodyor antigen is indicative of a past or chronic infection. For example, adecrease in the amount of antibody or antigen in the sample taken at thesecond time point (e.g., after treatment of the subject for a T.vaginalis infection) is indicative that the infection at the time thefirst sample was taken was an acute infection. Furthermore, if there isan increase in titer of antibody or amount of antigen, this wouldindicate an ongoing/active infection that was not diagnosed initially orthat was not eliminated upon diagnosis and drug treatment. This wouldnecessitate additional examination of body sites and tissues for thepresence of organism, antigen, or antibody.

Furthermore, a T. vaginalis protein of this disclosure can detect, butis not limited to, a recombinant α-enolase, aldolase, GAPDH and/orα-actinin protein as described in the EXAMPLES section set forth herein,as well as peptides of the reactive epitopes, fragments, andimmunologically-similar variants of such proteins, peptides andfragments. Such epitopes and recombinant proteins and peptides can beproduced according to methods well known in the art and can also beproduced by fractionation and/or isolation techniques, synthesistechniques, etc. that are known for producing proteins and peptides foruse in immunoassays.

The term “Trichomonas” as used herein, includes, but is not limited to aprotozoan parasite of the order Trichomonadida, genera Ditrichomonas,Trichomonas, Tritrichomonas and Pentatrichomonas, comprising multiplespecies that infects both humans and animals. “Trichomonas” refers toany Trichomonas species, e.g., Tritrichomonas foetus (also known asTrichomonas foetus, Tt. fetus), Tt enteris and T. paviovi, which infectcattle; Tt. suis, Tt. rotunda and T. buttreyi, which infect swine; Dt.ovis, which infects sheep; Tt. equi and T. equibuccalis, which infecthorses; T. anatis, Tt. eberthi, T. gallinae and T. gallinarum, whichinfect birds; Tt. caviae, Tt muris, Tt. wenoni, Tt. minuta and T.microti, which infect rodents; T. canistomae and T. felistomae, whichinfect dogs and cats; and T. tenax, T. vaginalis, Pt. hominis, and T.macacovaginae, which infect primates (including humans). Trichomonasvaginalis as described herein includes isolates T016, T068-II, UT40, andVB102, as well as any other T. vaginalis isolate now known or lateridentified.

The term “antibody” as used herein, includes, but is not limited to apolypeptide encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof. An antibody may be produced in a species otherthan the species of the subject putatively affected by a Trichomonasinfection. “Antibody” also includes, but is not limited to, apolypeptide encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof, which specifically binds to and recognizes theantigen-specific binding region (idiotype) of an antibody produced bythe host in response to exposure to T. vaginalis or other Trichomonasspecies antigen(s). Antibodies may also be produced using recombinantDNA gene engineering to generate synthetic linear or conformationalantibodies that recognize and bind to their cognate antigen(s).

The term “epitope” means an antigenic determinant that is specificallybound by an antibody. Epitopes usually consist of surface groupings ofmolecules, such as amino acids and/or sugar side chains, and may belinear or have specific three-dimensional structural characteristics, aswell as specific charge characteristics.

The term “15-mer epitope” and “15-mer amino acid sequences” are usedinterchangeably to describe the building blocks of a “series ofepitopes” (see definition of series of epitopes below). An epitopetypically comprises about 3 to 15 residues which are highly immunogenic.The epitope should have enough amino acid residues so that the peptideproduct is large enough to be recognized, which will generally be atleast 4-5 amino acids, but can be up to 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or at least 40 amino acids. The peptide 15-mers encodehighly immunogenic epitope regions (from a protein expressed by apathogenic microorganism of interest), having at one or both ends 1 to 7or more, e.g. 3 to 5, amino acids of naturally occurring sequence inorder to mimic the tertiary structure of protein folding to recapitulatethe native protein domain. These sequences are also called 15-merepitopes in order to distinguish them from the epitope within the nativeprotein. However, they could easily be made small or larger, generallywithin the range of 3 to 50 amino acids, 10 to 25 amino acids, or 12 to20 amino acids. Those of skill in the art will recognize that 15 aminoacids is considered to be a starting point or “default” size fordesigning short peptide sequences of epitopes. A 15-mer is thought to besufficiently large enough to allow correct folding and presentation ofan immunogenic site or protein domain, without having extraneous freeends that might hinder access to the site of interest. It can be easilyunderstood that a 14-mer, 16-mer, or any other oligopeptide of about3-50 amino acids could also be used in practicing the invention, so longas it comprises the essential core of the immunogenic amino acidsprovided in each sequence of the invention, shown in various tablesherein, and is functionally immunogenic.

The terms “series of epitopes” or “string of epitopes” (SOE),“recombinant series of epitopes” (rSOE), and “string of pearls” (SOP)are used interchangeably to refer to a synthetic macromolecule encodinga plurality of epitopes, e.g. two or more epitopes. The epitopes encodedin the SOE, rSOE, or SOP macromolecules may be peptide 15-mers (or15-mer amino acid sequences) comprising about 3 to 15 residues which arehighly immunogenic. In some embodiments, the epitopes comprise more than15 total residues. For example, a 16-mer to 40-mer epitope may includeabout 3 to 15 immunogenic residues. Selection of epitopes and/or 15-merepitopes to be included in a SOE is based specificity of the sequence,i.e., having no identity to other proteins in databases. This isespecially true with the SOEs that have epitopes and/or 15-mer epitopesfrom proteins expressed by other organisms. Thus, selecting uniquesequences helps to eliminate false positives that may occur due torecognition of proteins or antibodies to proteins from other organisms.The plurality of epitopes are typically arrayed in a linear moleculelinked with amino acid linkers. rSOE protein can be expressed in hostcells transfected or transformed with a vector carrying a nucleic acidencoding SOE or SOP sequences.

The SOE macromolecule may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more epitopes. For example, in some embodiments up to 100 or moreepitopes are included in the SOE macromolecule. As the size of the SOEmacromolecule increases, the isoelectric point (pI) becomes importantfor the stability of the SOE macromolecule. The isoelectric point is thepH at which the macromolecule carries no net electrical charge or iselectrically neutral in the statistical mean. In some embodiments, theSOE macromolecule has a pI from 3.00 to 7.00, from 4.50 to 5.50, or from4.90 to 5.10. In some embodiments, the pI is about 5.05. Whenconstructing such a SOE chimeric protein, the pI may be high (>9.0),which has high probability that the recombinant E. coli will place theprotein into inclusion bodies and be unrecoverable. Decreasing the pIthrough selection of amino acid linkers increases the probability thatE. coli will synthesize the protein in a soluble form for ease ofrecovery.

The epitopes in the SOE macromolecule may be connected by amino acidlinkers. The linkers may comprise 1 to 50 or more amino acids in anysequence. In some embodiments, the linker comprises repeats of 1, 2, 3,4, 5, or more amino acids. For example, the linker may comprise repeatsof any amino acid including, but not limited to, glycine (-GG-), lysine(-KK-), glutamic acid (-EE-), asparagine (-NN-), threonine (-TT-),alanine (-AA-), leucine (-LL-), arginine (-RR-), histidine (-HH-),aspartic acid (-DD-), serine (-SS-), glutamine (-QQ-), cysteine (-CC),proline (-PP-), valine (-VV-), isoleucine (-II-), methionine (-MM-),phenylalanine (-FF-), tyrosine (-YY-), tryptophan (-WW-), and mixturesthereof. The linkers within a single SOE macromolecule may be the sameor different. Selection of linker is based on the final pI of the SOEchimeric protein as well as hydrophobicity and antigenicity profilesthat affects solubility of the protein.

The term “highly immunogenic” means that the amino acids encoded by thesequences indicated will selectively and specifically bind to antibodiesraised against a particular sequence during a natural infection and/orimmunization. For example, the epitopes, 15-mer epitopes, and SOEs ofthe invention from regions of T. vaginalis α-actinin will detect thepresence of T. vaginalis antibodies in in vitro detection assays.Accordingly, antibodies raised against the epitopes, 15-mer epitopes,and SOEs of the invention from regions of T. vaginalis α-actinin willdetect the presence of T. vaginalis α-actinin protein or proteinfragments.

The terms “specifically binds to” and “specifically reactive with” referto a binding reaction that is determinative of the presence of theantigen and antibody in the presence of a heterogeneous population ofproteins and other biologics. Thus, under designated assay conditions,the specified antibodies and antigens bind to one another and do notbind in a significant amount to other components present in a sample.Specific binding to a target analyte under such conditions may require abinding moiety that is selected for its specificity for a particulartarget analyte. A variety of immunoassay formats may be used to selectantibodies specifically reactive with a particular antigen. For example,solid-phase enzyme-linked immunosorbent assays (ELISA) are routinelyused to select monoclonal antibodies specifically immunoreactive with ananalyte. See Harlow and Lane (ANTIBODIES: A LABORATORY MANUAL, ColdSprings Harbor Publications, New York, (1988)) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity. Typically a specific or selective reactionwill be at least twice background signal to noise and more typicallymore than 10 to 100 times greater than background.

An “immunologically reactive fragment” of a protein refers to a portionof the protein or peptide that is immunologically reactive with abinding partner, e.g., an antibody, which is immunologically reactivewith the protein itself.

As used herein, the term “antibody-antigen complex” can refer to animmune complex that forms when an antibody binds to its preferred orrecognized antigen. The antigen may be a full-length native protein, orit may be a protein fragment, either naturally occurring or synthetic.The antigen may further be an epitope, and that epitope may besynthetic. An antigen may further be a 15-mer epitope or an SOE, asdefined above. As disclosed herein, a discussion of antibody-epitopecomplex may further mean a complex of one or more 15-mer epitopes orSOEs and one or more antibody types. An immune complex comprising anantibody and an epitope may also be referred to as an antibody-epitopecomplex to distinguish it from an antibody-antigen complex, however,both antibody-epitope complexes and antibody-antigen complexes can becollectively referred to as immune complexes.

As used herein, the term “vaccine” refers to a composition that may beused to treat an individual or to provide protection against challenge,and more specifically it provides protection against a challenge mountedby exposure to or infection with a microorganism. For example, an SOEcomposed of an array of T. vaginalis epitopes in a solution suitable forinjection into a subject may provide protection from trichomoniasis. AnSOE comprising an array of N. gonorrohoeae epitopes may provideprotection from gonorrohea, and an SOE comprising an array of T.pallidum epitopes may provide protection from syphilis.

As used herein, the term “immunogenic composition” refers to acomposition comprising a SOE, rSOE, and/or SOP composed of epitopes thatelicit an immune response. For example, an SOE composed of an array ofT. vaginalis epitopes in a solution suitable for injection into asubject may elicit an immune response to T. vaginalis infection. An SOEcomprising an array of N. gonorrohoeae epitopes may elicit an immuneresponse to N. gonorrohoeae infection, and an SOE comprising an array ofT. pallidum epitopes may elicit an immune response to T. palliduminfection.

Antibodies to T. vaginalis proteins can be generated using methods thatare well known in the art. Such antibodies can include, but are notlimited to, polyclonal, monoclonal, chimeric, humanized, single chain,Fab fragments, and fragments produced by an expression library,including phage display. (See, e.g., Paul, FUNDAMENTAL IMMUNOLOGY, 3rdEd., 1993, Raven Press, New York, for antibody structure andterminology.)

Antibody fragments that contain specific binding sites for a T.vaginalis protein can also be generated. For example, such fragmentsinclude, but are not limited to, the F(ab′)₂ fragments that can beproduced by pepsin digestion of the antibody molecule, and the Fabfragments that can be generated by reducing the disulfide bridges of theF(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonalantibody Fab fragments with the desired specificity (Huse et al.,Science 254, 1275-1281 (1989)).

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionof chemically-stabilized whole organisms or any extract or lysate oforganisms comprising total proteins or with a T. vaginalis protein(e.g., individual or a combination of aldolase, GAPDH, α-enolase and/orα-actinin proteins) or any fragment or oligopeptide or conjugate thereofthat has immunogenic properties. One or more epitopes, 15-mer epitopesand/or SOEs may be used for injection into hosts for the production ofantibodies. Depending on the host species, various adjuvants can be usedto increase the immunological response. Such adjuvants include, but arenot limited to, Freund's complete and incomplete adjuvant, mineral gelssuch as aluminum hydroxide, and surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. Examples of adjuvants usedin humans include BCG (bacilli Calmette-Guerin) and Corynebacteriumparvum.

Monoclonal antibodies (MAbs) to Trichomonas vaginalis proteins can beprepared using any technique that provides for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (Kohler, et al.(1975) Nature 256:495-497; Kozbor, et al. (1985) J. Immunol. Methods81:31-42; Cote, et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole,et al. (1984) Mol. Cell Biol. 62:109-120). Briefly, the procedure is asfollows: an animal is immunized with a T. vaginalis protein, such asindividual or a combination of aldolase, GAPDH, α-enolase and/orα-actinin proteins, or immunogenic fragment or oligopeptide or conjugatethereof. For example, haptenic oligopeptides of a T. vaginalis proteincan be conjugated to a carrier protein to be used as an immunogen.Lymphoid cells (e.g., splenic lymphocytes) are then obtained from theimmunized animal and fused with immortal cells (e.g., myeloma orheteromyeloma) to produce hybrid cells. The hybrid cells are screened toidentify those that produce the desired antibody.

Human hybridomas that secrete human MAb can be produced by the Kohlerand Milstein technique. Although human antibodies are especiallypreferred for treatment of humans, in general, the generation of stablehuman-human hybridomas for long-term production of human MAb can bedifficult. Hybridoma production in rodents, especially mouse, is a verywell established procedure and thus, stable murine hybridomas provide anunlimited source of antibody of select characteristics. As analternative to human antibodies, the mouse antibodies can be convertedto chimeric murine/human antibodies by genetic engineering techniques.See Oi, et al., Bio Techniques 4(4):214-221 (1986); Sun, et al.,Hybridoma 5 (1986).

The MAbs of this invention specific for T. vaginalis protein epitopescan also be used to produce anti-idiotypic (paratope-specific)antibodies. (See e.g., McNamara et al., Science 220, 1325-26 (1984);Kennedy et al., Science 232:220 (1986).) These antibodies resemble theT. vaginalis protein epitope and thus can be used as an antigen tostimulate an immune response against the T. vaginalis protein.

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, et al., Proc. Natl. Acad.Sci. 81:6851-6855 (1984); Neuberger, et al., Nature 312:604-608 (1984);Takeda, et al., Nature 314:452-454 (1985)). Alternatively, techniquesdescribed for the production of single chain antibodies can be adapted,using methods known in the art, to produce T. vaginalis protein-specificsingle chain antibodies. Antibodies with related specificity, but ofdistinct idiotypic composition, can be generated by chain shuffling fromrandom combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad.Sci. 88:11120-3 (1991)).

Antibodies can also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as described in the literature(Orlandi, et al., Proc. Natl. Acad. Sci. 86:3833-3837 (1989)); Winter,et al., Nature 349:293-299 (1991)).

Various immunoassays can be used to identify antibodies of thisinvention having the desired specificity. Furthermore, a wide variety ofimmunoassays may be employed in the methods of this disclosure to detectantibodies and antigens of T. vaginalis proteins for diagnosis of T.vaginalis infection. Such immunoassays typically involve the measurementof antigen/antibody complex formation between a T. vaginalis protein orpeptide and its specific antibody.

The immunoassays of the invention can be either competitive ornoncompetitive. In competitive binding assays, T. vaginalis antigen orantibody competes with a detectably labeled T. vaginalis antigen orantibody for specific binding to a capture site bound to a solidsurface. The concentration of labeled antigen or antibody bound to thecapture agent is inversely proportional to the amount of free antigen orantibody present in the sample.

Noncompetitive assays can be, for example, sandwich assays, in which thesample analyte (target antibody) is bound between two analyte-specificbinding reagents. One of the binding agents is used as a capture agentand is bound to a solid surface. The other binding agent is labeled andis used to measure or detect the resultant antigen/antibody complex bye.g., visual or instrument means. A number of combinations of captureagent and labeled binding agent can be used. For instance, antigensderived from the T. vaginalis can be used as the capture agent andlabeled anti-human antibodies specific for the constant region of humanantibodies can be used as the labeled binding agent to detect antibodiesin a sample that bind the T. vaginalis antigen. Goat, sheep and othernon-human antibodies specific for human immunoglobulin constant regionsare well known in the art. Alternatively, the anti-human antibodies canbe the capture agent and the antigen can be labeled. Other proteinscapable of specifically binding human immunoglobulin constant regions,such as protein A, protein L or protein G can also be used as thecapture agent or labeled binding agent. These proteins are normalconstituents of the cell walls of streptococcal bacteria. They exhibit astrong non-immunogenic reactivity with immunoglobulin constant regionsfrom a variety of species. (See, e.g., Kronval, et al., J. Immunol.,111:1401-1406 (1973); Akerstrom, et al., J. Immunol., 135:2589-2542(1985).)

The non-competitive assays need not be sandwich assays. For instance,the antibodies or antigens in the sample can be bound directly to thesolid surface. The presence of antibodies or antigens in the sample canthen be detected using labeled antigen or antibody, respectively.

In some embodiments, antibodies and/or T. vaginalis protein or epitopesof proteins either singly or in combination of aldolase, GAPDH,α-enolase and/or α-actinin proteins, can be conjugated or otherwiselinked or connected (e.g., covalently or non-covalently) to a solidsupport (e.g., bead, plate, slide, dish, membrane or well) in accordancewith known techniques. Further, a plasmid construct encoding arecombinant protein that contains the epitopes of aldolase, GAPDH,α-enolase and/or α-actinin proteins detected by human antibodiesfollowing infection by and exposure to T. vaginalis can be used. Thisprotein comprised by a series of the epitope sequences is referred to asa SOE, rSOE, or SOP with each “pearl” representing an individualepitope, and the epitope can be separated by amino acid linkers, such asglycine (-GG-), lysine (-KK-), or glutamic acid (-EE-) repeats.Antibodies can also be conjugated or otherwise linked or connected todetectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵I, ³²P, ¹³H, ¹⁴C,¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), gold beads, chemiluminescence labels, ligands (e.g.,biotin) and/or fluorescence labels (e.g., fluorescein isothiocyanate) inaccordance with known techniques.

A variety of organic and inorganic polymers, both natural and syntheticcan be used as the material for the solid surface. Non-limiting examplesof polymers include polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon,nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF),silicones, polyformaldehyde, cellulose, cellulose acetate,nitrocellulose, and the like. Other materials that can be used include,but are not limited to, include paper, glass, ceramic, metal,metalloids, semiconductive materials, cements and the like. In addition,substances that form gels, such as proteins (e.g., gelatins),lipopolysaccharides, silicates, agarose and polyacrylamides can be used.Polymers that form several aqueous phases, such as dextrans,polyalkylene glycols or surfactants, such as phospholipids, long chain(12-24 carbon atoms) alkyl ammonium salts and the like are alsosuitable. Where the solid surface is porous, various pore sizes can beemployed depending upon the nature of the system.

A variety of immunoassay systems can be used, including but not limitedto, radio-immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA)assays, enzyme immunoassays (EIA), “sandwich” assays, gel diffusionprecipitation reactions, immunodiffusion assays, agglutination assays,immunofluorescence assays, fluorescence activated cell sorting (FACS)assays, immunohistochemical assays, protein A immunoassays, protein Gimmunoassays, protein L immunoassays, biotin/avidin assays,biotin/streptavidin assays, immunoelectrophoresis assays,precipitation/flocculation reactions, immunoblots (Western blot;dot/slot blot); immunodiffusion assays; liposome immunoassay,chemiluminescence assays, library screens, expression arrays, etc.,immunoprecipitation, competitive binding assays and immunohistochemicalstaining. These and other assays are described, among other places, inHampton et al. (Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn. (1990)) and Maddox, et al. (J. Exp. Med. 158:1211-1216(1993)).

The methods of this disclosure can also be carried out using a varietyof solid phase systems, such as described in U.S. Pat. No. 5,879,881, aswell as in a dry strip lateral flow system, such as described, forexample, in U.S. Patent Publication No. 20030073147, the entire contentsof each of which are incorporated by reference herein.

A subject is any animal that can be infected by Trichomonas vaginalis.In certain embodiments, the subject is human.

In addition, a nucleic acid (DNA) having the nucleotide sequence or asubstantially similar nucleotide sequence of the gene encoding the T.vaginalis protein of this disclosure can be used as a probe in a nucleicacid hybridization assay for the detection of a T. vaginalis protein invarious tissues or body fluids of a subject. Further, DNA encoding thesequence of the epitopes, 15-mer epitopes, and/or SOEs detected by humanserum following infection by and exposure to T. vaginalis can be used asa probe in a nucleic acid hybridization assay for the detection of a T.vaginalis protein in various tissues or body fluids of a subject. Theprobe can be used in any type of nucleic acid hybridization assayincluding Southern blots (Southern, 1975, J. Mol. Biol. 98:508),Northern blots (Thomas et al., 1980, Proc. Natl Acad. Sci. U.S.A.77:5201-05), colony blots (Grunstein, et al., 1975, Proc. Natl Acad.Sci. U.S.A. 72:3961-65), slot blots, dot blots, etc. Stringency ofhybridization can be varied depending on the requirements of the assayaccording to methods well known in the art. Assays for detecting nucleicacid encoding a T. vaginalis protein in a cell, or the amount thereof,typically involve, first contacting the cells or extracts of the cellscontaining nucleic acids therefrom with an oligonucleotide probe thatspecifically binds to nucleic acid encoding a T. vaginalis protein orpeptide as described herein (typically under conditions that permitaccess of the oligonucleotide to intracellular material), and thendetecting the presence or absence of binding of the oligonucleotideprobe thereto. Any suitable assay format can be employed (see, e.g.,U.S. Pat. Nos. 4,358,535; 4,302,204; 4,994,373; 4,486,539; 4,563,419;and 4,868,104, the disclosures of each of which are incorporated hereinby reference in their entireties).

The antibodies of this disclosure can be used in in vitro, in vivoand/or in in situ assays to detect a T. vaginalis protein or peptide ofthis disclosure.

Also as used herein, the terms peptide and polypeptide are used todescribe a chain of amino acids, which correspond to those encoded by anucleic acid (DNA). A peptide usually describes a chain of amino acidsof from two to about 30 amino acids and polypeptide usually describes achain of amino acids having more than about 30 amino acids. It isunderstood, however, that 30 is an arbitrary number with regard todistinguishing peptides and polypeptides and the terms may be usedinterchangeably for a chain of amino acids around 30. The peptides andpolypeptides of the present invention are obtained by isolation andpurification of the peptides and polypeptides from cells where they areproduced naturally or by expression of a recombinant and/or syntheticnucleic acid encoding the peptide or polypeptide. The peptides andpolypeptides of this invention can be obtained by chemical synthesis, byproteolytic cleavage of a polypeptide and/or by synthesis from nucleicacid encoding the peptide or polypeptide. The term polypeptide can referto a linear chain of amino acids or it can refer to a chain of aminoacids, which have been processed and folded into a functional protein.The term polypeptide can refer also the sequence of the epitopes. UsingT. vaginalis as an example, the selected epitopes from the proteins ofaldolase, GAPDH, α-enolase, and/or α-actinin are arranged so that eachepitope is separated by an amino acid linker, such as repeats ofglycine, lysine, or glutamic acid in the form of an SOE, rSOE, or SOP.

It is also understood that the peptides and polypeptides of thisdisclosure may also contain conservative substitutions where a naturallyoccurring amino acid is replaced by one having similar properties andwhich does not alter the function of the peptide or polypeptide. Suchconservative substitutions are well known in the art. Thus, it isunderstood that, where desired, modifications and changes may be made inthe nucleic acid and/or amino acid sequence of the peptides andpolypeptides of the present invention and still obtain a peptide orpolypeptide having like or otherwise desirable characteristics. Suchchanges may occur in natural isolates or may be synthetically introducedusing site-specific mutagenesis, the procedures for which, such asmismatch polymerase chain reaction (PCR), are well known in the art. Oneof skill in the art will also understand that polypeptides and nucleicacids that contain modified amino acids and nucleotides, respectively(e.g., to increase the half-life and/or the therapeutic efficacy of themolecule), can be used in the methods of the invention.

“Nucleic acid” as used herein refers to single- or double-strandedmolecules which may be DNA, comprised of the nucleotide bases A, T, Cand G, or RNA, comprised of the bases A, U (substitutes for T), C, andG. The nucleic acid may represent a coding strand or its complement.Nucleic acids may be identical in sequence to a sequence that isnaturally occurring or may include alternative codons that encode thesame amino acid as that which is found in the naturally occurringsequence. Furthermore, nucleic acids may include codons that representconservative substitutions of amino acids as are well known in the art.The nucleic acids of this invention can also comprise any nucleotideanalogs and/or derivatives as are well known in the art.

As used herein, the term “isolated nucleic acid” means a nucleic acidseparated or substantially free from at least some of the othercomponents of the naturally-occurring organism, for example, the cellstructural components commonly found associated with nucleic acids in acellular environment and/or other nucleic acids. The isolation ofnucleic acids can therefore be accomplished by well-known techniquessuch as cell lysis followed by phenol plus chloroform extraction,followed by ethanol precipitation of the nucleic acids. The nucleicacids of this disclosure can be isolated from cells according to methodswell known in the art for isolating nucleic acids. Alternatively, thenucleic acids can be synthesized according to standard protocols welldescribed in the literature for synthesizing nucleic acids.Modifications to the nucleic acids are also contemplated, provided thatthe essential structure and function of the peptide or polypeptideencoded by the nucleic acid are maintained.

The nucleic acid encoding the peptide or polypeptide of this disclosurecan be part of a recombinant nucleic acid construct comprising anycombination of restriction sites and/or functional elements as are wellknown in the art that facilitate molecular cloning and other recombinantDNA manipulations. Thus, the present disclosure further provides arecombinant nucleic acid construct comprising a nucleic acid encoding apeptide and/or polypeptide of this disclosure. The protein products ofcombinations of genetic sequences into a recombinant nucleic acid aresometimes referred to as chimeric proteins, polypeptides and/orpeptides, and the SOEs of the disclosure can be called such.

The present disclosure further provides a vector comprising a nucleicacid encoding a peptide and/or polypeptide of this disclosure. Thevector can be an expression vector which contains all of the geneticcomponents required for expression of the nucleic acid in cells intowhich the vector has been introduced, as are well known in the art. Theexpression vector can be a commercial expression vector or it can beconstructed in the laboratory according to standard molecular biologyprotocols. The expression vector can comprise, for example, viralnucleic acid including, but not limited to, vaccinia virus, adenovirus,retrovirus, alphavirus and/or adeno-associated virus nucleic acid. Thenucleic acid or vector can also be in a liposome or a delivery vehicle,which can be taken up by a cell via receptor-mediated or other type ofendocytosis.

The nucleic acid can be in a cell, which can be a cell expressing thenucleic acid whereby a peptide and/or polypeptide of this disclosure isproduced in the cell. In addition, the vector can be in a cell, whichcan be a cell expressing the nucleic acid of the vector whereby apeptide and/or polypeptide of this disclosure is produced in the cell.It is also contemplated that the nucleic acids and/or vectors can bepresent in a host (e.g., a bacterial cell, a cell line, a transgenicanimal, etc.) that can express the peptides and/or polypeptides of thepresent disclosure.

In some embodiments, for recombinant production of the chimericproteins, polypeptides and/or peptides of this disclosure inprokaryotes, there are numerous Escherichia coli (E. coli) expressionvectors known to one of ordinary skill in the art useful for theexpression of nucleic acid encoding proteins or peptides of thisinvention. Other microbial hosts suitable for use include bacilli, suchas Bacillus subtilis, and other enterobacteria, such as Salmonella,Shigella, and Serratia, as well as various Pseudomonas species. Theseprokaryotic hosts can support expression vectors that will typicallycontain sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence and have ribosomebinding site sequences for example, for initiating and completingtranscription and translation. If necessary, an amino terminalmethionine can be provided by insertion of a Met codon 5′ and in-framewith the coding sequence of the protein. Also, the carboxy-terminalextension of the protein can be removed using standard oligonucleotidemutagenesis procedures.

Additionally, yeast expression systems and baculovirus systems, whichare well known in the art, can be used to produce the chimeric peptidesand polypeptides of this disclosure.

The vectors of this disclosure can be transferred into a cell bywell-known methods, which vary depending on the type of cell host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, lipofection orelectroporation can be used for other cell hosts.

The present disclosure further provides a kit for detection ofmicroorganism-specific proteins. In the case of T. vaginalis, at leastone antibody is selected from the group consisting of aldolase, GAPDH,α-enolase and/or α-actinin antibodies, as disclosed in the sequencelistings and tables herein. Such a kit can comprise one or more proteinsor antibodies of the disclosure, along with suitable buffers, washsolutions, dilution buffers, secondary antibodies, and detectionreagents for the detection of antigen/antibody complex formation undervarious conditions. In another embodiment, a kit can comprise at leastone amino acid sequence selected from the group consisting of SOE,polypeptide, a peptide, and antigenic fragment comprising the amino acidsequence (epitope) detected by the monoclonal antibody and/or a fusionprotein or peptide comprising an individually or in combination theepitopes of interest, along with suitable buffers, wash solutions,dilution buffers, secondary antibodies, detection reagents, etc. for thedetection of antigen/antibody complex formation under variousconditions.

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the hundredth of the unit of the lower limitunless the context clearly dictates otherwise, between the upper andlower limit of that range and any other stated or intervening value inthat stated range, is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included inthe smaller ranges and are also encompassed within the invention,subject to any specifically excluded limit in the stated range. Wherethe stated range includes one or both of the limits, ranges excludingeither or both of those included limits are also included in theinvention.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

EXAMPLES

The present invention is more particularly described in the Examples setforth below, which are not intended to be limiting of the embodiments ofthis invention.

Example 1. Monoclonal Antibodies (MAbs) that Specifically Bind aTrichomonas vaginalis Fructose-1,6-Biphosphate Aldolase (Aldolase),glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), and/or α-EnolaseProteins

Detecting a T. vaginalis protein selected from aldolase, GAPDH,α-enolase and/or α-actinin proteins, in a sample, includes contacting asample with an antibody that specifically binds a T. vaginalis proteinor epitopes of proteins either singly or in combination selected fromthe group of aldolase, GAPDH, α-enolase and α-actinin proteins, underconditions whereby an antigen/antibody complex can form, and detectingformation of an antigen/antibody complex, thereby detecting the proteinin the sample. The method may be performed using an immunoassay, such asa dot blot, ELISA, or other high-throughput immunoassay, with ELISAbeing the preferred immunoassay.

In particular embodiments, the antibody employed in the methods of thisdisclosure is an antibody that specifically binds a T. vaginalis proteinor epitopes of the proteins either singly or in combination selectedfrom aldolase, GAPDH, α-enolase and/or α-actinin proteins. Anon-limiting example of an antibody that specifically binds the knownamino sequence of the epitope of a T. vaginalis protein selected fromaldolase, GAPDH, α-enolase and/or α-actinin proteins is monoclonalantibody ALDwsu1 (aldolase), ALDwsu2 (aldolase), GAPwsu2 (GAPDH),GAPwsu3 (GAPDH), ENOwsu2 (α-enolase), ENOwsu3 (α-enolase), ENOwsu4(α-enolase), ENOwsu6 (α-enolase) and HA423 (α-actinin).

A library of new monoclonal antibodies (MAbs) was generated toward theT. vaginalis proteins aldolase,glyceraldehyde-3-phosphate-dehydrogenase, and α-enolase proteins. Thenewly-generated MAbs are all IgG₁ isotype. MAbs B44, B43, and HA423 areincluded for comparative purposes and were generated at the Universityof Texas Health Science Center at San Antonio but the epitope amino acidsequences were unknown until now. All of the newly-generated MAbsreadily detect the surface of trichomonads as evidenced by wholecell-enzyme-linked immunosorbent assay (WC-ELISA) and fluorescence ofnon-permeabilized organisms. The respective proteins are detected byimmunoblot after SDS-PAGE blotting the proteins onto nitrocelluloseafter probing with individual MAbs. The amino acid sequences detected bythe respective MAbs (epitopes) are provided, and it is noteworthy thatthe epitopes detected by the MAbs generated at WSU are different fromthat B44 and B43 MAbs.

In certain embodiments, an antibody of this disclosure is notcross-reactive with human epithelial cell extracts or other protozoanprotein extracts (e.g., G. lamblia, E. histolytica, A. castellanii, L.major), fungi (Candida and Pneumocystis), and bacteria (oral and vaginalbacterial flora). In yet other embodiments, an antibody of thisdisclosure does not bind or react with T. vaginalis adhesin proteins.

TABLE 1 New MAbs generated and reactive with aldolase (ALD),GAPDH (GAP), α-enolase (ENO) and α-actinin. amino acid original WSU MAb,numbers in surface size protein name designation¹ proteinepitope sequence detection (kDa) SEQ ID NO: 1 ALD ALD12A ALDwsu-1142-149 RPDYVTVE yes 36.3 SEQ ID NO: 2 ALD ALD64A ALDwsu-2 166-173KLITYTRPE yes ″ SEQ ID NO: 3 ALD B44 N/A 448-455 ERIQKYTR yes 52SEQ ID NO: 4 ALD ALD11A ENOwsu-2 364-371 DDLYTTNP yes ″ SEQ ID NO: 5 ALDALD13B ENOwsu-3  7-14 AIVKECIA yes ″ SEQ ID NO: 6 ALD ALD25 ENOwsu-4463-470 LKEHDMLA yes ″ SEQ ID NO: 7 ENO ALD55 ENOwsu-6 64-71 YLGRVTLAyes ″ SEQ ID NO: 8 GAP B43 N/A 205-212 RRARAAGM yes 39.2 SEQ ID NO: 9GAP ALD30A GAPwsu-2 70-77 KSIGGRLG yes ″ SEQ ID NO: 10 GAP ALD32CGAPwsu-3 34-44 YLLKYDTAHRA yes ″ SEQ ID NO: 11 ACT HA423 N/A 649-653YKVTY yes 106.2Detection of Fixed Trichomonas vaginalis Protein in Fixed CellPreparations.

Pap smears were prepared using methanol (MeOH) as a fixative with MeOHin the range of 20% to 80%. Trichomonads are readily fixed by incubationin MeOH prepared in PBS buffer and retain integrity as visualized bydarkfield microscopy. Surface-exposed epitopes are readily detected byMAbs as shown in Table 1.

The following ELISA protocol used for pap smear was used to showimmuno-detection of protein on MeOH-fixed trichomonads immobilized ontowells of microtiter plates.

-   1. Overnight (o/n) cultures of T. vaginalis grown using standard    protocols in medium were washed twice with ice-cold PBS.-   2. Trichomonads were fixed o/n at 4° C. (12 h to 18 h) using 20%    MeOH (different concentrations of MeOH yield similar results) at a    density of 10⁷ per milliliter (ml).-   3. 100 microliters (μl) of different dilutions of parasites in MeOH    fixative were added to individual wells of 96-well microtiter    plates, and plates were placed in 37° C. incubator overnight.-   4. To dried wells was added 100 μl of a solution of 1% BSA in PBS-1%    Tween (PBS-T) and incubated at 37° C. for 30 min.-   5. Wells were then washed 3-times (3×) with PBS-T followed by    addition of 100 μl of each MAb (primary antibody) to wells. Negative    controls included PBS (absence of primary MAb) and the addition of    MAb L64, which detects a small-sized (17-kDa) cytoplasmic protein.    (NOTE: This MAb of the same isotype (IgG₁) does not detect the    parasite surface showing the integrity of parasites by fixation.)    Wells were incubated for 60 min at 37° C.-   6. After washing 3× with PBS-T, 100 μl of a solution of secondary    horseradish peroxidase-conjugated goat anti-mouse IgG antibody was    added to wells followed by incubation at 37° C. for 30 min.-   7. The wells were again washed 3× with PBS-T prior to addition of    100 μl of color development reagent. After 15 min, the microtiter    well plates were read for intensity of optical density at 405 nm    wavelength to measure absorbance values. A higher the absorbance    value at 405 nm indicates strong immunoreactivity by MAb with the    protein antigen on the surface of MeOH-fixed parasites.    Results are shown in FIGS. 1 and 2. Among noteworthy findings are    the following:    -   a) Sensitivity was ≥1 organism per μl of sample. Wells were        incubated with 100 μl of the parasite suspension ≥10³/ml. At the        lowest density, this is equal to 100 organisms added to each        well.    -   b) All trichomonad isolates are readily detected.    -   c) Parasite preparations made with a poly-bacterial        contamination does not interfere with MAb detection.    -   d) The presence of epithelial cells contaminating trichomonad        preparations does not interfere with MAb detection.    -   e) Newly-generated MAbs give higher signal compared to the HA423        MAb patented to the University of Texas system.    -   f) Protein was detected using the range of MeOH for fixation.    -   g) The signal was enhanced when a cocktail of three MAbs, one        each for aldolase, GAPDH, and α-enolase, was used.

Example 2. Detection of Trichomonas vaginalis Proteins and Antibodies inSaliva

T. vaginalis is a urogenital, mucosal parasite. Presently, there existsa point-of-care, antigen detection, lateral flow, immunochromagraphicdiagnostic that is used for women. This diagnostic is not useful fordiagnosis in men. This diagnostic is invasive for women, because itrequires obtaining a vaginal swab. Therefore, there is a need for adifferent, non-invasive diagnostic that will work for both men andwomen.

Both female and male patients make specific anti-trichomonad surfaceprotein IgG antibody. This antibody is detectable in serum and vaginalwashes in women and serum in males. Antibody at one mucosal site makesit possible to detect the antibody at distant mucosal sites. Patientsare known to make antibody to the proteins aldolase, GAPDH, α-enolaseand α-actinin, making these proteins candidates for detection by saliva.Therefore, a diagnostic based on saliva antibody that detects theseproteins or epitopes of the proteins either singly or in combinationimmobilized on a platform represents a diagnostic that is used for bothfemale and male patients.

The availability of the MAbs to these proteins permits purification ofrecombinant proteins from cDNA expression libraries or by purificationby MAb-affinity column chromatography. Alternatively, the epitopes knownto be reactive by sera of both women and men represent targets that canbe synthesized and immobilized for detection by saliva. Therefore, theindividual or combination of aldolase, GAPDH, and α-enolase proteins orthe combination of reactive epitopes of the proteins aldolase, GAPDH,and α-enolase are reagents used in a non-invasive, oral-saliva baseddiagnostic. Finally, the polypeptide either synthesized or derived fromrecombinant DNA that possess the sequence of the epitopes in theproteins of aldolase, GAPDH, α-enolase, and/or α-actinin whereby eachepitope is separated by such amino acids as glycine, lysine, or glutamicacid (SOE) is a reagent used in a non-invasive, oral-saliva baseddiagnostic.

Patient saliva has antibody specific to whole cell T. vaginalis and totrichomonad proteins of aldolase, GAPDH, α-enolase, and/or α-actinin. Awhole cell-ELISA was carried out, in which microtiter wells were coatedwith whole T. vaginalis cells. Saliva of individual T.vaginalis-infected patients and pooled saliva of healthy, uninfectedindividuals were then tested for reactive IgG using horse radishperoxidase-conjugated anti-human IgG secondary antibody. Each patientshows elevated absorbance values compared to the control pooled salivaof uninfected individuals. This study demonstrates the presence of IgGantibody reactive to whole T. vaginalis proteins. Wells coated withwhole cells tested separately using rabbit anti-T. vaginalis serum orwith a MAb served as positive controls and were also used forstandardization to show similar reactions among wells. Non-reactiveserum of men and women and prebleed normal rabbit serum served asnegative controls.

In separate experiments, the individual sera of women and men highlyreactive with the whole cell-ELISA and with each of the aldolase, GAPDH,α-enolase, and α-actinin proteins above were each reacted withoverlapping, synthetically-made dodecapeptides comprising the entireamino acid sequence of the aldolase, GAPDH, α-enolase proteins andα-actinin proteins. The overlapping dodecapeptides were spotted(immobilized) onto a membrane that was then probed individually with 10%dilution of highly WC-ELISA reactive sera of women or men. The seradetected all of the epitopes to which antibody was present. Nododecapeptides were detected by negative control, unreactive sera ofwomen and men. This study demonstrates the existence andimmunoreactivity of sera of both women and men to various epitopes andalso demonstrated that the women and men sera detected the same epitopesrecognized by the MAbs included in Table 1 to the aldolase, GAPDH,α-enolase and α-actinin proteins.

In yet another experiment, the individual sera of women and men highlyreactive with T. vaginalis organisms and with each of the aldolase,GAPDH, α-enolase, and α-actinin proteins above were each reacted withsynthetic 15-mer peptides possessing the epitopes of the aldolase,GAPDH, α-enolase, and α-actinin proteins either singly or in combinationwere immobilized onto a membrane that was then probed individually with10% dilution of highly WC-ELISA reactive sera of women or men. The seradetected all of the epitopes to epitopes singly and in combination. Nopeptides singly or in combination were detected by negative control,unreactive sera of women and men. This study demonstrates the existenceand immunoreactivity of sera of both women and men to various epitopesand also demonstrated that the women and men sera detected the sameepitopes recognized by the MAbs included in Table 1 to the aldolase,GAPDH, α-enolase and α-actinin proteins.

In yet another experiment, the individual sera of women and men highlyreactive with T. vaginalis organisms and with each of the aldolase,GAPDH, α-enolase, and α-actinin proteins above were each reacted with arecombinant polypeptide possessing the epitopes of the aldolase, GAPDH,α-enolase, and α-actinin proteins (SOE) immobilized onto a membrane thatwas then probed individually with 10% dilution of highly WC-ELISAreactive sera of women or men. The sera detected all of the epitopesthis recombinant polypeptide possessing the epitopes of the aldolase,GAPDH, α-enolase, and α-actinin proteins. No peptides singly or incombination were detected by negative control, unreactive sera of womenand men. This study demonstrates the existence and immunoreactivity ofsera of both women and men to various epitopes and also demonstratedthat the women and men sera detected the same epitopes recognized by theMAbs included in Table 1 to the aldolase, GAPDH, α-enolase and α-actininproteins.

No crossreactivity of saliva antibody between T. vaginalis and theopportunistic oral T. tenax. Saliva of humans uninfected with T.vaginalis has no detectable antibody using any of the ELISA assaysmentioned above. Thus, the existence of immunocrossreactive antibodiesin saliva of patients to T. tenax, the oral trichomonad will benon-existent. T. tenax organisms are not readily apparent in the oralcavity and are not detectable in individuals even if there is severeperiodontitis.

Demonstration of specific anti-T. vaginalis antibody in saliva ofpatients. Standard ELISA can demonstrate the existence of salivaantibody in all patients. The assays can be optimized to minimize anycrossreactive antibody to T. tenax and to monitor the level of salivaantibody among the patients, although, as just mentioned above, there isno evidence of salivary antibody crossreactivity with T. tenax. Threedifferent assays provide a basis by which to determine the level ofantibody to trichomonad proteins in saliva. ELISA protocols that bindnon-specific sites on the coated wells with irrelevant proteins, such asBSA and/or or skim milk, can be employed. The first ELISA has wholeintact trichomonads coated onto 96-well microtiter plates as antigen forsaliva antibody detection, and this whole cell-ELISA employs standardconditions. For this whole cell ELISA, MeOH-fixed trichomonads can beused, or, alternatively, PBS-washed organisms can be added to wells andallowed to dry overnight. Then ethanol is added to the dried wells, andwells allowed to dry and fix the trichomonads onto the wells. The secondELISA has purified IgG of high-titered rabbit antisera to totaltrichomonad proteins coated onto microtiter wells. Then, trichomonadprotein antigens from a detergent extract of T. vaginalis will bind tothe IgG-coated wells after incubation. The bound trichomonad proteinsprovide antigen detectable by saliva antibody. Similarly, the thirdassay has a cocktail of MAbs to aldolase, GAPDH, α-enolase andα-actinin-coated onto microtiter wells. These MAbs-coated wells bindprotein antigen from the trichomonal extract. These parasite proteinsbound to MAbs will now serve as antigen for saliva antibody. The secondand third sandwich-ELISAs take advantage of the knowledge that women andmen make serum antibody to various epitopes of each protein (Table 1).It is expected that saliva antibody, like serum antibody, is directed toepitopes different from those of rabbit antiserum and that we have nowshown are detected by serum antibody of women and men infected andexposed to T. vaginalis.

After treatment of freshly prepared ELISA plates with skim milk todecrease non-specific interactions, select samples of saliva frompatients and from uninfected control individuals can be diluted in PBSwithout or with a T. tenax detergent extract prior to addition ofstandard 100 μl volumes to microtiter wells. PBS without T. tenaxextract provides duplicates of the same saliva. Initial data shows thatsaliva does not have any antibody to T. tenax, and data suggests thatany concern of crossreactivity is nonexistent. Experiments indicate thata 60 min to 120 min incubation at 37° C. is optimal. After washing,horseradish peroxidase-conjugated secondary goat anti-human IgG,anti-IgA, or Ig (IgG+IgA+IgM) Ab is added, followed by color developmentwith substrate. In these assays, purified trichomonad protein calledP230 that is a prominent immunogen eliciting a vaginal IgG antibodyresponse will serve as a positive control for saliva IgG antibody.

Saliva antibody from women infected with T. vaginalis and aftertreatment is tested. Saliva can be obtained on at least two occasionspost-treatment to assess the nature of the antibody response followingremoval of trichomonads from the urogenital tract through drugtreatment. Saliva from male partners of infected women can also beexamined to confirm the validity of this diagnostic for both infectedpartners.

Example 3. Trichomonas vaginalis Proteins Detected in Urine by MAbs

The numerous proteins that are increased in expression during infection,found in secretions of patients, and are readily secreted bytrichomonads have been identified and include aldolase, GAPDH, α-enolaseand α-actinin. Both women and men patients infected with T. vaginalishave trichomonads in urine. This means that many proteins and/or intactorganisms may be detected in urine samples for both women and men. Amongthe many secreted proteins found in large amounts are those to which thenew MAbs have been generated, and these proteins are aldolase, GAPDH,and/or α-enolase. These proteins are readily detected by immunoblotswith the MAbs to aldolase, GAPDH, α-enolase and/or α-actinin. Therefore,these proteins in soluble form in urine can be immobilized throughfiltration and can then be detected by the MAbs. Although the presentinvention has been described with reference to specific details ofcertain embodiments thereof, it is not intended that such details shouldbe regarded as limitations upon the scope of the invention except as andto the extent that they are included in the accompanying claims.Throughout this application, various patents, patent publications andnon-patent publications are referenced. The disclosures of thesepatents, patent publications, and non-patent publications in theirentireties are incorporated by reference into this application in orderto more fully describe the state of the art to which this inventionpertains.

Overview of Examples 4 Through 7: Identification of DiagnosticImmunogenic Epitopes of Aldolase, GAPDH, α-Enolase, and α-Actinin thatare Reactive with Sera of Female and Male Patients and MonoclonalAntibodies (MAbs) and are Unique to Trichomonas vaginalis

We have identified epitopes of the T. vaginalis proteins aldolase,GAPDH, α-enolase, and α-actinin with little or no identity to othersexually transmitted microorganisms [Treponema pallidum (syphilis),Chlamydia trachomatis (chlamydia), Neisseria gonorrhoeae (gonorrhea),and Candida albicans (yeast)], normal flora bacteria (E. coli), yeast(Saccharomyces cerevisiae), and humans (Homo sapiens) and are,therefore, unique targets to diagnose infection and exposure to T.vaginalis. These peptide epitopes have significance for diagnosis ofinfection with T. vaginalis. The experimental approach likewiseidentified epitopes in the trichomonad GAPDH and α-enolase proteins withsignificant identity to peptide epitopes in the human proteins to whichindividuals infected with T. vaginalis make antibody. Such antibodyduring a T. vaginalis infection may have consequences for autoimmunity.The GAPDH peptide epitopes were found to have high sequence identity tothe GAPDH protein of Tritrichomonas suis parasite of porcine, which is asynonym for Tritrichomonas foetus-bovine, the causative agent of fetalwastage in cattle, Tritrichomonas foetus-cat, causative agent forchronic large-bowel diarrhea, and Tritrichomonas mobilensis, entericprotozoan of squirrel monkeys (Lun, Z.-R., et al., Trends in Parasitol.,21:122-125, 2005; Reinmann, K., et al., Veterinary Parasitol., publishedahead of print, doi: 10.1016/j.vetpar.2011.09.032.). Therefore, theseepitope characterization experiments have identified diagnostic epitopesof the important pathogenic porcine, cattle, cat, and squirrel monkeytrichomonads (Tritrichomonas suis, T. foetus-bovine, T. foetus-cat, andT. mobilensis).

Overlapping dodecapeptides of each of the aldolase, GAPDH, α-enolase andα-actinin proteins were examined for immunoreactivity with the sera ofwomen and men patients and MAbs. The overlapping dodecapeptides for eachof the proteins were immobilized in succeeding spots on a template thatpermitted detection by antibodies. This procedure is standard foridentification of epitopes immunogenic during infection or that reactwith serum antibody and MAbs. This approach permits analysis of theantibody responses that are similar and distinct between the sera ofwomen and men patients in addition to localizing the epitopes detectedby MAbs. Further, it is possible to perform comparative analysis of theamino acid sequences of similar functional proteins of humans (Homosapiens), of other sexually transmitted bacterial pathogens (Neisseriagonorrhoeae, Treponema pallidum subsp. pallidum strain Nichols, andChlamydia trachomatis), of yeast (Saccharomyces cerevisiae and Candidaalbicans), and of other human bacterial pathogens (Streptococcuspyogenes, Streptococcus pneumoniae, and Staphylococcus aureus).Alignment of the amino acid sequences reveals whether the peptidesequences are unique to Trichomonas vaginalis, are identical and commonto other Trichomonas sp. (T. suis and synonyms T. foetus-bovine, T.foetus-cat, and T. mobilensis), and share high or identical sequenceidentity with humans (H. sapiens) that may have significance forautoimmune reactions.

Accompanying each of the following experiments are tables showing thediagnostic immunogenic sequences reactive with female and male sera andMAbs that are unique to T. vaginalis. Other tables illustrate the extentof sequence identity between the T. vaginalis amino acid sequences withthose of other bacteria, yeast, and human. These alignments wereobtained from BLAST amino acid sequence alignments of proteins. Spotnumbers on the overlapping peptides and the numbers of amino acids inthe T. vaginalis peptide epitopes reactive with female (F) and male (M)sera and MAbs and that are unique to T. vaginalis are provided under thecolumn labeled “unique Tv epitope for diagnosis” and given a positive(+) sign. T. vaginalis peptide epitopes with high sequence identity toTritrichomonas suis (synonym with T. foetus-bovine, T. foetus-cat, andT. mobilensis) protein epitope sequences are also disclosed. Thesepeptides are reactive with female or male sera or both, and illustratetheir utility also for diagnosis of porcine, cattle, cat, and squirrelmonkey trichomonads. The peptides of the proteins α-enolase and GAPDHwith high sequence identity to human protein epitopes and with possibleautoimmune crossreactivity are listed, and the T. vaginalis (Tv) peptidesequences are aligned with the human sequences (Hu).

Example 4. Identification of Diagnostic Immunogenic Epitopes of AldolaseUnique to T. vaginalis

TABLE 2 Identification of diagnostic immunogenicepitopes of fructose-1,6-biphosphate aldolase protein that are unique toT. vaginalis (Tv). female male no. amino patient patient acid epitopesera sera Mab unique sequence sequence reactivity reactivity reactivityTo Tv SEQ ID NO: 12 40-47 AIITASVK F1 SEQ ID NO: 13 58-65 AGARKYAN M1SEQ ID NO: 14 61-71 RKYANQTMLRY F2 SEQ ID NO: 15  91-101 PIVLHLDHGDS F3SEQ ID NO: 1 142-149 RPDYVTVE ALD12A SEQ ID NO: 2 166-173 KHYTYTRPEALD64A + SEQ ID NO: 16 169-179 YTRPEEVQDFV F4 SEQ ID NO: 17 193-203TSHGAYKFPPG F5 SEQ ID NO: 18 231-241 SIPQEYVEMVN F6 SEQ ID NO: 19277-287 RMVMTGTIRRL M2 SEQ ID NO: 20 298-305 RQYLGEAR F7 M3SEQ ID NO: 21 304-311 ARTKLTEM F8 +The range of percent identity of these peptide epitopes with selectedpathogens and humans is illustrated in the sequence alignment data ofFIGS. 3A, 3B, and 3C. Abbreviations F and M refer to female and malepatient sera reactive with corresponding epitopes. The plus (+) signrefers to the epitope sequences that are unique to T. vaginalis, asevidenced by absence of sequence identity shown in FIGS. 3A, 3B, and 3C.Sequence Alignment of the T. vaginalis Fructose-1,6-BisphosphateAldolase (ALD) Protein with ALD Proteins of Other RepresentativeOrganisms and H. sapiens.FIGS. 3A, 3B, and 3C shows the amino acid sequence comparison of T.vaginalis (Tv, SEQ ID NO:147) ALD with the homolog proteins fromTreponema pallidum (Tp, SEQ ID NO:148), Neisseria gonorrhoeae (Ng, SEQID NO:149), Streptococcus pyogenes (Spy, SEQ ID NO:150), Streptococcuspneumoniae (Spn, SEQ ID NO:151), Staphylococcus aureus (Sa, SEQ IDNO:152), Escherichia coli. Candida albicans, Saccharomyces cerevisiae,and Homo sapiens. The boxed amino acids contained in the T. vaginalissequence are the epitopes presented in Table 2. The order from top tobottom of the sequences is based on from highest to lowest percentidentity compared to T. vaginalis ALD sequence. All microorganismsrepresented are pathogens, and 15-mer epitopes and/or SOEs derived fromthe ALD or other proteins may be used to practice the invention.

TABLE 3 Aldolase percent epitope sequence identity comparisons withbacterial, yeast, and human sequences from alignment shown in FIGS. 3A,3B, and 3C. ALD12A and ALD64A are MAbs. Epitope Organism F1 M1 F2 F3ALD12A ALD64A F4 F5 F6 M2 F7 M3 F8 T. vaginalis 100 100 100 100 100 100100 100 100 100 100 100 T. pallidum 62.5 87 72.7 100 87 50 72.7 63.654.5 54.5 75 37.5 N. 37.5 87.5 54.5 63.6 37.5 0 28.57 72.7 45.4 45.4 500 gonorrhoeae S. pyogenes 37.5 44.4 16.7 54.5 37.5 0 14.3 27.3 0 9.1 250 S. 37.5 44.4 25 54.5 25.00 0 14.29 27.3 0 9.1 25 0 pneumoniae S.aureus 50 33.3 8.3 63.6 37.5 25 45.4 45.4 0 9.1 75 37.5 E. coli 25 33.316.7 63.6 25 25 41.7 45.4 0 9.1 25 12.5 C. albicans 25 33.3 16.7 54.512.5 50 35.7 36.4 0 18.2 25 0 S. cerevisiae 25 33.3 16.7 54.5 12.5 5035.7 27.3 0 18.2 25 0 Homo 0 12.5 9.1 27.3 0 0 0 9 0 9.1 12.5 0 sapiensHydrophobicity and Antigenicity Profiles of the ALD, ENO, and GAPSequences.FIG. 4 presents analyses of hydrophobicity and antigenicity alignmentsin reference to epitopes along the protein. Of interest is that with fewexceptions the epitopes represent hydrophilic regions contained withinthe protein, perhaps consistent with presentation of amino acids forantibody synthesis and recognition.

Based on the features of the epitopes, 7 epitopes for ALD, 8 for ENO,and 6 for GAP were selected for synthesis of 15-mer peptides encoding inan SOE. The individual amino acid sequence encoding the epitope is boldand underlined.

Example 5. Identification of Diagnostic Immunogenic Epitopes ofα-Enolase Unique to Trichomonas vaginalis

TABLE 4 Identification of diagnostic immunogenicepitopes of α-enolase protein unique toT. vaginalis (Tv). Epitope amino acid sequencesunique to the T. vaginalis protein are based onpercent identity shown in Table 5. no. female male SEQ amino patientpatient ID acid epitope sera sera MAb unique NO: sequence sequencereactivity reactivity reactivity to Tv 22  6-14 AIVKECIA ALD13A + 2364-71 YLGRVTLA F1 ALD55 + 24 70-77 LAARSSAP M1 + 25  94-101 DKARYGGK F2M2 26 139-146 TVLKKNIG F3 M3 + 27 184-194 VPKKFKLPSPF F4 M4 + 28 238-246GGLLVKKY F5 + 29 245-252 KYGLSAKN M7 + 30 298-305 FYDEEKKL M8 + 31328-338 KKHPAIVSIED F6 32 343-362 ENWTKLNARLG F7 33 364-371 DDLYTTNPALD11A 34 448-455 ERIQKYTR F9 M11 B44 35 463-471 LKEHDMLA ALD25 +

TABLE 5 The extent of sequence identity of the T. vaginalis α-enolasewith the protein sequences of bacteria, yeasts, and human. The epitopesare indicated by F1 through F9, M1 through M7, and the MAbs ALD13A/B,ALD55, ALD11A, and ALD25 are as listed in the Table. Epitope F1 F2 F3 F4Organism ALD13A ALD55 M1 M2 M3 M4 F5 T. vaginalis 100 100 100 100 100100 100 T. pallidum 0.00 0.00 25 71.4 37.5 27.3 37.5 N. 0 0 25 71.4 5027.3 25 gonorrhoeae C. 0 0 12.5 57.1 37.5 27.3 12.5 trachomatis S.pyogenes 0 0 25 71.4 37.5 27.3 12.5 S. 0 0 25 85.7 37.5 27.3 12.5pneumoniae S. aureus 0 0 25 71.4 37.5 27.3 25 E. coli 0 0 25 57.1 37.536.4 25 C. albicans 0 12.5 50 42.8 37.5 54.5 50 S. cerevisiae 0 0 37.542.8 37.5 36.4 37.5 Homo 0 12.5 37.5 71.4 37.5 27.3 37.5 sapiens EpitopeF9 F6 M7 Organism M5 M6 F7 F8 ALD11A B44 ALD25 T. vaginalis 100 100 100100 100 100 100 T. pallidum 0 25 63.6 27.3 50 37.5 5.5 N. 12.5 25 54.545.4 75 37 10.5 gonorrhoeae C. 0 37.5 54.5 45.4 62.5 75 0 trachomatis S.pyogenes 12.5 37.5 54.5 45.4 50 50 10.5 S. 12.5 37.5 54.5 45.4 50 5010.5 pneumoniae S. aureus 12.5 25 54.5 36.4 62.5 50 10.5 E. coli 25 2572.7 18.3 62.5 37.5 10 C. albicans 75 37.5 63.6 27.3 75 37.5 6.3 S.cerevisiae 62.5 25 73.7 27.3 75 37.5 6.3 Homo 62.5 37.5 54.5 36.4 75 506.3 sapiens Abbreviations: F, female antibody reacting with epitope; M,male antibody reacting with epitope; ALD refers to MAbs.

TABLE 6 Identification of epitopes ofTrichomonas vaginalis (Tv) α-enolase reactive with human sera.¹ % spotspooled identity number amino female reactive with on acid epitopepatient male MAb unique human membrane sequence sequence sera serareactions to Tv sequence SEQ ID NO: 22   2.3  6-14 AIVKECIA ALD13A +SEQ ID NO: 23 21-23 64-71 YLGRVTLA F1 ALD55 + SEQ ID NO: 24 23-25 70-77LAARSSAP M1 + SEQ ID NO: 25 31-33  94-101 DKARYGGK F2 M2 71%SEQ ID NO: 26 46-47 139-146 TVLKKNIG F3 M3 + SEQ ID NO: 27  62   184-194VPKKFKLPSPF F4 M4 + SEQ ID NO: 135  67   199-209 NGGKHAGGNLK M5SEQ ID NO: 136  76   226-236 OLRMVAEVYQK M6 SEQ ID NO: 28 79-81 238-746GGLLVKKY F5 + SEQ ID NO: 29 81-82 245-252 KYGLSAKN M7 62.5%SEQ ID NO: 30  99-100 298-305 FYDEEKKL M8 + SEQ ID NO: 31 110   328-338KKHPAIVSIED F6 + SEQ ID NO: 32 115-118 343-362 ENWTKLNARLG F7 +SEQ ID NO: 137 117-118 352-359 NARLGQRV M9 SEQ ID NO: 33 121-123 364-371DDLYTTNP ALD11A 75% SEQ ID NO: 138 123-124 370-377 NPITIKKG F8 M10SEQ ID NO: 34 149-151 448-455 ERIQKYTR F9 M11 B44 + SEQ ID NO: 35154-155 463-471 LKEHDMLA ALD25 +

Example 6. Identification of Diagnostic Immunogenic Epitopes of GAPDHUnique to Trichomonas vaginalis, and Diagnostic Epitopes Identical to orwith High Sequence Identity to Tritrichomonas suis (Synonym with T.foetus Bovine, T. foetus Cat, and T. mobilensis)

TABLE 7 Identification of diagnostic immunogenicepitopes of GAPDH protein unique to T. vaginalis. no. female male aminopatient patient acid epitope sera sera MAb unique sequence sequencereactivity reactivity reactivity to Tv SEQ ID NO: 36 13-17 LYPKD M1 +SEQ ID NO: 37 34-44 YLLKYDTAHRA F1 ALD32C + SEQ ID NO: 38 58-68FTVGEGADKWV M2 + SEQ ID NO: 39 70-77 KSIGGRLG F2 ALD30A + SEQ ID NO: 40 94-101 STGIFRTK F3 + SEQ ID NO: 41 106-113 AEGKIKKD F4 + SEQ ID NO: 42118-125 HLVSGAKK F5 M4 SEQ ID NO: 43 157-161 SNASC M5 SEQ ID NO: 44175-182 NAEGIRNG F6 + SEQ ID NO: 45 205-912 RRARAAGM F7 B43SEQ ID NO: 46 217-274 TSTGAAIA F8 SEQ ID NO: 47 229-233 CHGLP M6SEQ ID NO: 48 250-260 SLVDLTVNVNA F9 SEQ ID NO: 49 292-299 VSSDIIGC M7SEQ ID NO: 50 298-305 GCQYSSIV M8 SEQ ID NO: 51 301-311 YSSIVDALSTK F10SEQ ID NO: 52 325-335 VSWYDNEWMY F11 M9 SEQ ID NO: 53 337-341 CRCAD M10

TABLE 8 The extent of sequence identity of the T. vaginalis GAPDH withthe protein sequences of bacteria, yeasts, and human from alignmentshown in FIG. 3. Epitope F1 F2 F5 F7 F11 Organism M1 ALD32 M2 ALD30 M3F3 F4 M4 M5 F6 B43 F8 M6 F9 M7 M8 F10 M9 M10 Overall T. vaginalis 100100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100100 T. suis 80 55 73 63 100 75 38 75 100 75 88 100 20 64 88 63 73 100100 70 C. albicans 0 45 9 25 40 50 13 56 100 50 38 75 20 55 50 63 45 6440 40 S. cerevisiae 0 45 9 13 60 50 13 56 100 63 38 75 20 61 63 50 45 6440 39 C. 0 55 0 0 60 50 13 67 100 50 38 75 20 55 88 75 55 55 40 38trachomatis T. pallidum 40 45 9 44 100 38 0 56 100 38 50 63 0 45 50 6355 82 40 40 N. 0 45 9 13 60 38 13 78 100 38 50 75 20 45 38 13 45 64 2041 gonorrhoeae S. 0 55 9 25 40 38 25 67 80 38 63 75 20 18 75 38 55 73 040 pneumoniae S. pyogenes 0 55 9 25 20 38 25 67 80 63 63 75 20 27 75 3855 73 0 40 S. aureus 0 45 0 25 60 38 13 56 100 75 50 88 20 55 38 25 5573 40 40 E. coli 0 55 9 13 40 38 0 56 100 50 38 75 20 55 50 25 27 64 2040 Homo 0 45 0 0 60 38 0 33 36 50 38 75 20 45 50 50 45 64 40 30 sapiens

TABLE 9 Identification of epitopes ofTrichomonas vaginalis (Tv) GAPDH reactive withhuman sera and MAbs and with identity to human and T. suis. % % identityidentity pooled unique Tv with with  SPOTS amino female reactive epitopehuman T. suis peptide acid epitope patient male MAb for peptide peptideno. sequence sequence serum sera reactions diagnosis sequence sequenceSEQ ID NO: 36 3-5 13-17 LYPKD M1 +  80 SEQ ID NO: 37  12 34-44YLLKYDTAHRA F1 ALD32C +  55 SEQ ID NO: 38  20 58-68 FTVGEGADKWV M2 +  73SEQ ID NO: 39 73-74 70-77 KSIGGRLG F2 ALD30A +  63 SEQ ID NO: 139 25-2779-83 SQLPW M3 60 100 SEQ ID NO: 40 31-33  94-101 STGIERTK F3 +  75SEQ ID NO: 41 35-37 106-113 AEGKIKKD F4 +  25 SEQ ID NO: 42 39-41118-125 HLVSGAKK F5 M4  75 SEQ ID NO: 43 51-53 157-161 SNASC M5 50 100SEQ ID NO: 44 57-59 175-182 NAEGIRNG F6 50  75 SEQ ID NO: 45 67-69205-212 RRARAAGM F7 B43  88 SEQ ID NO: 46 72-74 217-224 TSTGAAIA F8 75100 SEQ ID NO: 47 75-77 229-233 CHGLP M6 +  20 SEQ ID NO: 48  84 250-260SLVDLTVNVNA F9 +  64 SEQ ID NO: 49 97-98 292-299 VSSDIIGC M7 50  88SEQ ID NO: 50  99-100 298-305 GCQYSSIV M8 50  63 SEQ ID NO: 51 101301-311 YSSIVDALSTK F10 +  73 SEQ ID NO: 52 109 325-335 VLSWYDNEWMY F11M9 64 100 SEQ ID NO: 53 111-113 337-341 CRCAD M10 + 100

TABLE 10 Identification of T. vaginalis (Tv)GAPDH peptide epitopes that have high sequenceidentity to GAPDH of and are diagnosticfor Tritrichomonas suis (Ts). Only peptideepitopes of T. vaginalis with ≥50% identity were selected. no. amino(Tv) (Ts) Ts acid epitope SEQ epitope percent sequence sequence ID NO:sequence identity SEQ ID NO: 36 13-17 LYPKD 54 LYPKE  80   SEQ ID NO: 3734-44 YLLKYDTAHRA 55 HLLNYDSAHQR  55   SEQ ID NO: 38 58-68 FTVGEGADKWV56 FEVGTGSDKWV  73   SEQ ID NO: 39 70-77 KSIGGRLG 57 KNLTGRLG  62.5SEQ ID NO: 40  94-101 STGIFRTK 58 STGLFRTH  75   SEQ ID NO: 41 118-125HLVSGAKK 59 HLLAGAKK  75   SEQ ID NO: 43 157-161 SNASC 60 SNASC 100  SEQ ID NO: 140 172-179 TLNNAFGI 61 VLNDTEGI  57   SEQ ID NO: 141 202-212KDLRRARAAGM 62 RRARAAGM 100   SEQ ID NO: 142 247-257 ITGSLVDLTVN 63ITGSLVDITVN  91   SEQ ID NO: 51 301-311 YSSIVDALSTKV 64 HSSIVDSLSTMV 75   SEQ ID NO: 143 322-329 LVKVLSWY 65 LVKVLSWY 100  

Additional noteworthy evidence of the GAPDH crossreactivity between T.vaginalis and T. suis (T. foetus) is evidenced by data obtained bydetection on nitrocellulose of the T. foetus GAPDH after SDS-PAGE andimmunoblotting of total proteins of different T. foetus isolates. TheMAbs generated to the T. vaginalis GAPDH (Table 1) were used as probesto detect the T. foetus protein.

Example 7. Identification of Diagnostic Immunogenic Epitopes ofα-Actinin Unique to Trichomonas vaginalis

TABLE 11 Identification of diagnostic immunogenicepitopes of α-actinin protein unique to T. vaginalis (Tv) amino womanreactive SPOTS acid epitope patient men unique numbers sequence namesequence serum serum MAb to Tv SEQ ID NO: 66  2  4-14 ACT1 RREGLLDDAWEF1 + SEQ ID NO: 67 11-12 34-41 ACT2 IQFETIET F2 + SEQ ID NO: 68 21-2367-71 ACT3 KQPKM F3 + SEQ ID NO: 69 50 148-158 ACT4 YEHVAVNNFTT F4 +SEQ ID NO: 70 69 205-215 ACT5 YVYLDPEDVID F5 + SEQ ID NO: 71 80-82244-248 ACT6 ADKIK F6 + SEQ ID NO: 72 97-99 295-302 ACT7 RGKLASVI F7 +SEQ ID NO: 73 111-112 334-341 ACT8 NRPIPEIP F8 + SEQ ID NO: 74 152-153457-464 ACT9 HHSQLITY F9 M1 + SEQ ID NO: 75 165-166 496-503 ACT10YDEAIAFK F10 M2 + SEQ ID NO: 76 214-216 646-650 ACT11 KLNYK F11 M3 +SEQ ID NO: 77 215-217 649-653 ACT12 YKVTY F12 M4 HA423 + SEQ ID NO: 78268-270 808-812 ACT13 KYFDK F13 M5 +

TABLE 12 The extent of sequence identity of the T. vaginalis α-actininwith the protein sequences of bacteria, yeasts, and human. Epitope T.suis C. albicans S. cerevisiae HuACTN1 ACT-F1 (W1) 9 9 11 21 ACT-F2 (W2)0 0 0 50 ACT-F3 (W3) 0 20 20 40 ACT-F4 (W4) 9 18 9 46 ACT-F5 (W5) 0 18 036 ACT-F6 (W6) 14 14 20 40 ACT-F7 (W7) 0 0 13 25 ACT-F8 (W8) 0 0 25 38ACT-F1/M1 (W9/M1) 11 22 11 13 ACT-F10/M2 (W10/M2) 0 0 13 13 ACT-F11/M3(W11/M3) 20 0 20 20 ACT-F12/M4 20 20 20 0 (W12/M4/HA423) ACT-F13/M5(W13/M5) 0 20 0 40Note absence of sequence identity with all proteins for representativeorganisms shown in Table 12. There is no identity of epitopes with otherproteins of bacteria, fungi, protists, and humans in databanks.

Example 8. Synthesis of 15-Mer Peptide Epitopes of ALD, ENO, GAP, andACT Unique to Trichomonas vaginalis to Demonstrate Immunoreactivity withWomen and Men Sera

Based examination of the sequences with algorithms for hydrophobicityand antigenicity, we then selected 7 epitopes for ALD, 8 for ENO, and 6for GAP, and the 13 for ACT (taken from Table 11) for synthesis of15-mer peptides encoding the epitopes. Table 13 shows the epitopes andincludes 3 for ACT to which data are presented below. The individualamino acid sequence encoding the epitope within each 15-mer peptide isshown in bold and underlined.

Peptide epitopes from each protein were selected based on low percentidentity and solubility. The 15-mer amino acid sequences were sent toSigma-Aldrich (The Woodlands, Tex.) and synthesized using their CustomPepScreen Peptide service. Each individual 15-mer peptide contained wasacetylated at the amino-terminus and was amidylated at thecarboxy-terminus. Each 15-mer peptide was screened by mass spectrometryto determine yield and purity of each product. Peptide epitopes werereceived with a pass/fail designation and the amount provided. Threeα-actinin 15-mer peptides of ACT were used as positive controls in theexperiments presented below. These peptides were designated ACT2, ACT3,and ACT1 and corresponded to the amino acid sequences

(SEQ ID NO: 101) AQPLYDEAIAFKEEV, (SEQ ID NO: 102) FKDTFKYFDKDKSNS and(SEQ ID NO: 100) SVNRHHSQLITYIKH,respectively (shown in Table 13).

TABLE 13 List of synthetic 15-mer peptides ofrepresentative epitopes of ALD, ENO, GAP, andACT for reactivity with immunoreactive women and men sera. women/menamino epitope peptide acid amino acid designation name numbers sequenceSEQ ID NO: 79 A-W1 ALD1 36-50 EQLQ AIITASVK TES SEQ ID NO: 80 A-ALD12ALD2 138-152 EAHS RPDYVTVE GEL SEQ ID NO: 81 A-ALD64 ALD3 159-173EDDVKAE KHTYTRPE SEQ ID NO: 82 A-W4 ALD4 167-181 HT YTRPEEVQDFV SKSEQ ID NO: 83 A-W6 ALD5 230-244 SS SIPQEYVEMVN KY SEQ ID NO: 84 A-M2ALD6 275-289 DG RMVMTGTIRRL FV SEQ ID NO: 85 A-W8 ALD7 301-315 LGEARTKLTEM YMRK SEQ ID NO: 86 E-W1 ENO1 57-71 VK YLGRVTLA ARSSASEQ ID NO: 87 E-M1 ENO2 70-84 VT LAARSSAP SGAST SEQ ID NO: 88 E-W3,E-M3ENO3 136-150 TDG TVLKKNIG GNAC SEQ ID NO: 89 E-F4/M4 ENO4 182-196 DKVPKKFKLPSPF FN SEQ ID NO: 90 E-W5 ENO5 236-250 KL GGLLVKKY GLSAKSEQ ID NO: 91 E-M8 ENO6 295-309 SSE FYDEEKKL YEVE SEQ ID NO: 92 E-W7/M9ENO7 344-358 DY ENWTKLNARLG QR SEQ ID NO: 93 E-W8,E-M10 ENO8 366-380LYTT NPITIKKG LEG SEQ ID NO: 94 G-M1 GAP1  8-97 RACRK LYPKD IQVVASEQ ID NO: 95 G-M2 GAP2 56-70 QE FTVGEGADKWV VK SEQ ID NO: 96 G-W2 GAP367-81 WVV KSIGGRLG PSQL SEQ ID NO: 97 G-W4 GAP4 104-118 KD AEGKIKKDDGYDGH SEQ ID NO: 98 G-M6 GAP5 224-238 ALPKV CHGLP PKSLD SEQ ID NO: 99G-M10 GAP6 332-346 EWMYS CRCAD IFHRL SEQ ID NO: 100 ACT-W9,M1 ACT1463-467 SVNR HHSQLITY IKH SEQ ID NO: 101 ACT-W10,M2 ACT2 492-506 AQPLYDEAIAFK EEV SEQ ID NO: 102 ACT-W13,M5 ACT3 803-817 FKDTF KYFDK DKSNS

Approximately 1 μg of individual and/or a combination of syntheticpeptides were dot-blotted onto a nitrocellulose membrane and allowed toair dry for 30 min at 37° C. These dot-blots were fit into individualwells of a 96-well microtiter ELISA plate. Then, 100 μl of 2%ELISA-grade BSA (Sigma-Aldrich, St. Louis, Mo.) in PBS (eBSA-PBS), pH7.4, was added and incubated for 2 hours at RT, after which 5 μl of a1:1 dilution (v/v) of T. vaginalis negative- and positive-control womenor men sera in PBS, pH 7.4, was added and incubated for 30 min at RT.The remainder of the procedure is as detailed above. Densitometric scanswere produced using the ImageJ software (rsbweb.nih.gov/ij).

FIGS. 5A, 5B, 5C, and 5D present results from representative dot-blotreactions using positive control sera of women and men of 15-merpeptides for ALD (1a and 1b), ENO (2a and 2b), GAP (3a and 3b), and ACT(4a and 4b) as a positive control (XX). Reactivity was detected for each15-mer peptide, albeit at different levels of spot intensities. Nopeptides were detectable using negative control sera for both women andmen that were determined to be unreactive with the full-length proteins.These data suggest that some peptide-epitopes may be used asserodiagnostic targets.

We next performed dot-blots (FIG. 6A) using random combinations of the15-mer peptides to determine whether an increased extent of reactivitywas seen for women and men sera. We further wanted combinations ofpeptides that might give equal reactivity for both sera. FIG. 6Bpresents in duplicate the intensity of signal for each combination withthe positive control women and men sera (labeled 4+/5+), and nodetection of the peptide cocktails was evident with negative controlsera (labeled -0-). Densitometric scan (bars 1 through 7) revealedoverall better extent of reactions with combination of peptides comparedto the individual peptides for both women and men sera (data not shown).The combined peptides ACT2 and ACT3 (bars 8) served as positive controlswith known sera of men and women reactive with α-actinin, and the pooledpeptides GAP1, GAP6 and ENO3 (bars 9) as well as GAP5, ENO5, and ALD6(bars 10) showed no reactivity with negative control sera of both womenand men sera.

Example 8. Identification of Immunogenic Epitopes of α-Enolase and GAPDHwith High Sequence Identity to Human Protein Sequences

From Tables 6 and 9 it was possible to identify three epitopes ofα-enolase and one epitope of GAPDH with 62.5% to 78% identity to thehuman peptide sequences, as illustrated in Table 14. The peptidesequences were considered high identity if there was a difference insequence by only two to three amino acids in an eight to nine linearamino acid sequence.

TABLE 14 Identification of Trichomonas vaginalis (Tv)peptide epitopes with high sequence identity tohuman (Hu) protein epitopes and with possible immune crossreactivity.female male amino patient patient acid epitope sera sera MAb identityprotein sequence sequence reaction reaction reactivity (%) SEQ ID ENO 94-101 (Tv) DKARYGGK + + 78   NO: 25 SEQ ID (Hu) DKQRYLGK NO: 103SEQ ID ENO 245-252 (Tv) KYGLSAKN + 62.5 NO: 29 SEQ ID (Hu) KYGKDATNNO: 104 SEQ ID ENO 364-371 (Tv) DDLYTTNP + 75   NO: 4 SEQ ID(Hu) DDLTVTNP NO: 105 SEQ ID GAP 322-329 (Tv) LVKVLSWY + + 62.5 NO: 144SEQ ID (Hu) FVKLISWY NO: 106

Example 9. Production of Recombinant Protein Encoding Sequential EpitopeSequences of Aldolase, GAPDH, α-Enolase, and α-Actinin Separated byAmino Acid Spacers for Use in Serodiagnosis

In this example, the epitopes identified in Tables 2-11 that areimmunoreactive with seropositive sera of women and men for the proteinsaldolase, GAPDH, α-enolase, and α-actinin are encoded within a plasmidconstruct so that the individual epitopes are within 15-mer peptides ofthe trichomonad protein. The epitopes may be in any random order sothat, for example, the sequence of epitopes may include one for aldolasefollowed by one for α-actinin followed by one for α-enolase followed byone for aldolase, etcetera. Further, the number of epitopes may be justone each representative of each protein or as many as deemed necessaryfor each protein for optimal antibody detection in a serodiagnostic. Theplasmid construct is then expressed in recombinant E. coli, and arecombinant protein is then made upon induction. This type ofrecombinant protein containing a series or an array of epitopes isreferred to here as a String of Pearls (SOP) where each “pearl” isrepresentative of the amino acid sequence within which is found anepitope as described. This recombinant protein can also be referred tointerchangeably as a SOE or rSOE.

FIG. 7A presents a simple example of an SOP encoding sequentially twoepitopes of GAPDH, two epitopes of α-enolase, and two epitopes ofaldolase. The sequences of these individual epitopes and 15-mer epitopesare among those listed in Table 13 above. The SOP sequence identity isalso provided in SEQ ID NO:145. This and other recombinant SOP arrays ofepitopes of the T. vaginalis proteins aldolase, GAPDH, α-enolase, andα-actinin purified similarly can then be immobilized on surfaces forprobing and detection by immunoreactive sera of women and men. FIG. 7Bshows purification of an SOP, with lysate, flow-through, washes, andelution fractions in SDS-PAGE gel with Coomassie-brilliant blue stain.

The SOP encoding for an array of six 15-mer amino acid sequences, twoeach of which contained epitopes for ALD, ENO, and GAP was 111 aminoacids for an Mr of 13.35-kDa. The DNA encoding for the SOP with a His₆tag at the carboxy terminus was cloned into a pET23b expression plasmidconstruct that was transformed into E. coli B121DE3. Recombinant E. coli(rE. coli) was stored as glycerol stocks at −70° C. until used, whichwere thawed and streaked onto Luria Broth (LB) agar plates containing 25μg ampicillin (amp). Isolated colonies were inoculated into 200 ml freshLB containing amp and incubated in a shaker incubator at 37° C. and 220rpm. Following overnight growth, rE. coli were inoculated into fresh LBmedium with amp and incubated for 3 hours at 37° C. at 220 rpm prior toaddition of 1 mM IPTG and incubation an additional 3 hours. The rE. coliwere centrifuged using a Sorvall SLA-1500 rotor at 8,000 rpm and 4° C.for 15 minutes. Supernatant was decanted, and the pellet was stored at−80° C. until used. At various time intervals, prior to and after IPTGaddition, 1 ml of rE. coli were microfuged at 10,000 rpm for 15 minutesand pellets prepared for SDS-PAGE (34, 35) for analysis of recombinantSOP::His₆ fusion protein (12.2-kDa) expression after IPTG addition.Immunoblot analysis after SDS-PAGE, shown in FIG. 8, confirmed thesynthesis of SOP::His₆ using as probe positive control sera of women andmen that were seropositive to α-actinin as defined above.

ELISA was performed by immobilizing purified SOP protein onto 96-well,flat-bottom Nunc polysterene plates. Each well was coated with 100 μlcontaining 1 μg of SOP diluted in carbonate:bicarbonate buffer, pH 9.6,and the plates were incubated overnight at RT with gentle agitation.Each plate was then washed 3× with PBS-T. On the third wash the plateswere incubated in PBS-T for 5 minutes at RT with gentle agitation priorto removing the PBS-T. The plates were then incubated upside down o/n onat RT on paper towels before being covered with plastic wrap and storedat 4° C. until used. For testing, plates were washed twice with PBS-T.On the second wash the plates were incubated in PBS-T at RT for 5minutes with gentle agitation and slap-dried. Each well was then blockedwith 200 μl of eBSA-PBS for 2 hours at 37° C. Plates were then washedtwice with PBS-T. On the second wash the plates were incubated in PBS-Tat RT for 5 minutes with gentle agitation followed by removing thePBS-T. Next, 100 μl of a 1:25 dilution in eBSA-PBS of women and men serawas added to each well in duplicate and incubated at RT for 5 minuteswith gentle agitation before incubation for 4 hours at 37° C. The plateswere washed three times with PBS-T. On the third wash the plates wereincubated in PBS-T for 5 minutes at RT with gentle agitation. Afterremoval of the PBS-T, 100 μl of secondary horseradishperoxidase-conjugated goat-anti-human IgG (Fc-specific) diluted 1:1,500in eBSA-PBS was added to each well and incubated at RT with gentleagitation for 5 minutes before incubation for 1 hour at 37° C. Theplates were washed 3× with PBS-T, as above, prior to addition of 100 μlof color development solution, prepared according to the manufacturer'sinstructions well and incubated at RT with gentle agitation for 15minutes. Absorbance values at 405-nm were obtained using Bio-Tek platereader (Bio-Tek Instruments, Inc).

We performed assays to assess whether this novel recombinant protein isdetectable with positive control sera of women and men, as above. FIG.8A shows representative reactions by ELISA with SOP arrays immobilizedonto individual wells of 96-well microtiter plates. Negative controlsera of women and men had little to no reactivity as evidenced by lowA_(405 nm) with the SOP in comparison to the strong signals (highA_(405 nm)) obtained with positive sera of women and men. The SOP wasundetectable when using an irrelevant MAb HA423 to α-actinin of T.vaginalis (XX) compared to the very strong reaction seen with a positivecontrol MAb to hexa-histidine in the fusion recombinant protein.

Further, FIGS. 8B and 8C demonstrates that antibodies in positive seraof women and men react with the SOP array and are detected by dot-blots(FIG. 8B) and by immunoblot after SDS-PAGE and immunoblotting ontonitrocellulose (FIG. 8C). The Coomassie-brilliant blue stained gel is aduplicate from the SDS-PAGE used for immunoblotting of the SOP, and thePonceau S-stained nitrocellulose is a duplicate included to show thetransfer of the SOP protein onto nitrocellulose.

Example 9. Epitopes of Highly Immunogenic T. vaginalis α-Actinin Used asSerodiagnostic Targets for Both Women and Men

Highly immunogenic α-actinin protein and protein fragments werecharacterized to further establish utility as a target for serodiagnosisof trichomonosis for both women and men. It is known that the sera ofwomen with trichomonosis possess antibody reactive with numeroustrichomonad proteins, including α-actinin (referred to as positivecontrol women sera). Epitope mapping identified 13 peptide epitopeswithin α-actinin reactive to the positive control sera of women. Mensera highly-seropositive to the trichomonad parent α-actinin and thetruncated version called ACT-P2 (positive control men sera) identified 5epitopes that were a subset of those detected by positive control womensera. The amino acid sequences of the epitopes had little or no sequenceidentity to the human α-actinin homolog and to proteins of othermicrobial pathogens, including a related Tritrichomonas suis and yeastsCandida albicans and Saccharomyces cerevisiae. Further, immobilized15-mer peptides of representative epitopes are reactive to both positivecontrol women and men sera.

A plasmid was constructed to encode an SOP array of all thirteenepitopes of α-actinin as shown in FIG. 9 (SEQ ID NO:146). This actininSOP is expressed in E. coli as is the SOP presented above expressingepitopes of ALD, ENO, and GAP. In this case, the thirteen epitopesdetected antibodies in the sera of women and men exposed to T. vaginalisare arranged sequentially within individual 15-mer peptides, which wereseparated from each other by a diglycine (-GG-). recombinant E. coliwith the plasmid construct with the DNA sequence encoding thisrecombinant SOP of α-actinin is induced for expression of the protein.The α-actinin epitope protein is a fusion protein with a hexa-histidinesequence at the carboxy-terminus for purification as above bynickel-affinity chromatography. Purified α-actinin SOP can beimmobilized for detection antibodies in the sera of women and men.

Materials and Methods

α-Actinin-P2 (ACT-P2) Expression and Purification.

The natural T. vaginalis α-actinin protein consists of 931-amino acidsand is 106.2-kDa. This full-length highly immunogenic protein is usedfor examining the relation between seropositivity in men and prostatecancer. Subclones of the trichomonad α-actinin gene are made todetermine the region of the protein most reactive with men sera. Thissubclone encoded a protein of 558-amino acid protein from the aminoterminus, called ACT-P2. The coding region of ACT-P2 corresponding toamino acids 375 to 932 is PCR amplified and cloned in pET23b expressionvector with the kanamycin resistance gene (Kan^(r)) for transformationof E. coli BL21DE3 cells. The resulting recombinant 558-amino acidsequence further comprises a C-terminal His₆ tag fusion protein of63.5-kDa. Bacteria are grown on Luria Broth (LB) agar plates containing25 μg/ml Kan, and rE. coli is incubated for 3 hours at 37° C. at 220 rpmprior to addition of 1 mM isopropylthiogalactoside and incubated for anadditional 3 hours. The rE. coli are centrifuged using a SorvallSLA-1500 rotor at 8,000 rpm and 4° C. for 15 minutes. and pellets storedat −80° C. until used. Synthesis of ACT-P2 is confirmed using as probethe murine monoclonal antibody (MAb) HA423 (27-29) to trichomonadα-actinin or MAb to His₆ (Advanced Targeting Systems, San Diego, Calif.,USA).

For purification of ACT-P2, pellets of rE. coli are thawed for 15 min onice and suspended in 10-ml lysis buffer (50 mM Tris, pH 8.0, 300 mMNaCl, 10 mM β-mercaptoethanol (β-ME), and 0.1% Triton-X100), and lysatesare sonicated 10 times each at room temperature (RT) for 30 seconds(sec). Sonicates are centrifuged using a Sorvall SS-34 rotor at 8,000rpm and 4° C. for 20 minutes (min), and supernatant is applied to aNi²⁺-NTA superflow affinity column according to the manufacturer'sinstructions (Qiagen Inc., Valencia, Calif., USA). Purified ACT-P2protein is confirmed by SDS-PAGE and immunoblot using as probe MAbHA423, as above.

The Human ACTN1 Homolog.

The purified full-length human ACTN1 α-actinin homolog used in thisstudy is the isoform B protein of 892 amino acids (˜103-kDa) (NovusBiologicals, Littleton, Colo., USA). The soluble protein is in 74 mMTris-HCl, pH 8.0, containing 10 mM reduced glutathione. For ELISA andimmunoblot assays 1 μg of ACT-P2 and ACTN1 is used. ELISA is performedusing wells of microtiter plates coated with ACT-P2 or ACTN1 as detailedbelow. SDS-PAGE for immunoblotting onto nitrocellulose for both ACT-P2and ACTN1 is carried out using 7.5% acrylamide gels, as before (36, 37).

Positive Control Sera of Women and Men and Detection of Antibody toACT-P2.

During the course of our research on T. vaginalis we have examined˜1,000 sera of women patients with trichomonosis and, more recently, upto 20,000 sera of men for seropositivity to trichomonad proteins andparticularly α-actinin. We, therefore, were able to determine the extentof serum antibody to total T. vaginalis proteins, α-actinin, and ACT-P2by ELISA (27-29). Individual, α-actinin-seropositive sera of women andmen had identical or very similar reactivities to trichomonad proteinsand α-actinin. This permitted us to pool the sera to have sufficientamounts for conducting epitope mapping experiments, as outlined below,and was considered positive control sera. Likewise, pooled seronegativesera of both women and men were considered negative control sera forparallel experiments conducted throughout.

Trichomonad Natural α-Actinin SPOTs Membrane Synthesis for EpitopeMapping.

Oligopeptides derived from the sequences of T. vaginalis α-actinin(GenBank accession number AAC72899) were synthesized on activatedmembranes using the SPOTs system (Sigma-Genosys, The Woodlands, Tex.,USA). Five to 10 nmol of each peptide was covalently bound to a Whatman50 cellulose support (Whatman, Maidstone, England) by the C-terminususing Fmoc-L amino acid chemistry and had an acetylated N-terminus. Theoligopeptides were 11-mer amino acids in length and had a sequentialoverlap of eight amino acids. The SPOTs spanned the entire sequence ofthe protein.

Probing the α-Actinin SPOTs Membrane with Positive and Negative ControlSera and MAb HA423.

The membrane was initially washed with a small volume of 100% MeOH for 5minutes to avoid precipitation of hydrophobic peptides during thefollowing procedure. After washing 3× each for 10 minutes in 25 ml ofTBS buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, and 2.7 mM KCl), theSPOTs membrane was incubated in Blocking Buffer (TBS containing 5% BSA)at RT for 2 hours. The membrane was incubated with a 1:10 dilution ofnegative or positive control sera of women and men, respectively, andincubated overnight at 4° C. The membrane was also probed with the MAbHA423 that detects α-actinin. After washing 3× each for 5 minutes eachin TBS, the membrane was incubated with a 1:1,500 dilution of secondaryanti-human antibody as above or anti-mouse IgG Fab (IgG fraction)prepared in Blocking Buffer. After washing 3× each in TBS at RT for 5minutes each, bound antibodies were detected using color developmentreagent.

Immediately following color development and SPOT analysis, the membranewas regenerated by washing 3× with water with each wash for 10 minutesat RT with agitation. Bound antibody was stripped from the membrane bywashing at least 4 times with each wash for 30 minutes with RegenerationBuffer I (62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM ME) at 50° C.with agitation. The membrane was washed three times each for 20 minuteswith 10×PBS at RT with agitation, after which the membrane was washed 3×each for 20 minutes with T-TBS buffer (TBS, pH 8.0, containing 0.05%Tween 20) at RT with agitation. This was followed by washing 3× each for10 minutes with TBS at RT with agitation. The presence of any visiblespots resulted in repeating the regeneration steps. As a control to showthat the primary antibody was completely removed, the membrane wasre-incubated with the appropriate secondary antibody and substratesolution and developed. Regeneration was continued until no reactivitywas seen with secondary antibody.

The epitope amino acid sequences were determined based on reactivitiesof overlapping peptides, as shown above in Table 11. Epitope sequenceswere compared with other proteins by using the protein-protein basiclocal alignment search tool found on a page of the NCBI website. Aminoacid sequence alignments of the proteins were performed with CLC ProteinWorkbench (Muehltal, Germany). Hydrophobicity plots and antigenicityplots were constructed using Lasergene MegAlign (DNASTAR, Madison,Wis.).

Synthesis and Reactivity of Individual α-Actinin Epitopes.

Three 15-mer peptide epitopes identified from SPOT membrane epitopemapping with low percent identity to other human pathogens as well asthe α-actinin human homolog were synthesized in PEPscreen format(Sigma-Genosys). The reactivity of each peptide was tested withrepresentative negative and positive control sera either individually orin combination. Approximately 10 μg of peptide was blotted ontonitrocellulose membranes and air dried overnight at RT. The epitopeblots were then blocked with 2% e-BSA in PBS at 37 C for 2 hoursfollowed by incubation with 1:25 dilution in PBS of negative or positivecontrol women and men sera for ACT-P2 and incubated overnight at RT.This was followed by secondary antibody and color development, as above.All assays were performed in duplicate and repeated at least threetimes.

Results

Positive Control Sera of Women and Men does not Detect the Humanα-Actinin Homolog Protein.

Negative and positive control sera of women and men were used to probeimmunoblots of ACT-P2. As can be seen in FIG. 10 positive (pos) controlsera of women (lanes 3a and 3b) and of men (lanes 5a, and 5b) detectedACT-P2 in two separate experiments done at different times underidentical experimental conditions. Negative (neg) control sera of womenand of men gave no detectable bands (lanes 2 and 4, respectively). TheIgG₁ MAb HA423 to the trichomonad α-actinin used as probe gave strongreactivity in separate experiments (lanes 1 and 6), and, notunexpectedly, an irrelevant IgG₁ MAb B44 reactive with trichomonadα-enolase as a negative control detected no protein band.

We next tested for immuno-crossreactivity purified,commercially-available human α-actinin (HuACTN1) with pooled positivecontrol sera of women and men used in FIG. 10. FIG. 11A (lane 5) showsthe intense stained band of 1 μg HuACTN1. Gels with the same amount ofHuACTN1 were transferred onto nitrocellulose for immunoblotting. Aduplicate blot with HuACTN1 was stained to insure transfer of theprotein (not shown). The HuACTN1 was neither detected by the positivecontrol sera (lane 4) nor HA423 (not shown). As above, 1 μg of ACT-P2(lane 3) was readily detected by MAb HA423 (lane 1) and rabbit antiserumto total T. vaginalis proteins (IRS, lane2). We then tested byimmunoblot using total proteins of T. vaginalis (lane 10), as before,whether the pooled positive control sera of both women and men detectednumerous other trichomonad proteins. FIG. 11B shows that pooled negativecontrol sera did not detect any trichomonad proteins by immunoblot (lane7) whereas pooled positive control sera recognized numerous proteins(lane 8). As a control and not surprisingly, many trichomonad proteinsin the total protein preparation were evident when blots were probedwith IRS (lane 9). As with negative control sera of women and men (lane7), no proteins were detected by control, prebleed normal rabbit serum.Finally, we tested for detection of non-denatured HuACTN1 coated ontowells of microtiter plates. Again, neither positive control sera ofwomen and men nor MAb HA423 reacted to the HuACTN1-coated wells (notshown).

α-Actinin Epitopes React with Positive Control Sera of Women and Men andMAb HA423.

We next tested the positive control sera of women and men stronglyreactive with ACT-P2 for IgG antibody to overlapping 11-mer peptides ona Custom SPOTs membrane (Materials and Methods). Table 5A of Example 7lists the 13 epitopes and corresponding amino acid sequences labeled W1through W13 recognized by women sera. M1 through M5 represent the subsetof epitopes detected by men sera. The IgG₁ MAb HA423 detected the sameepitope as W12/M4. Negative control sera of women and men and the MAbsB43 and B44 to trichomonad GAPDH and α-enolase, respectively, which werethe same isotype as HA423, were unreactive with the SPOTs membrane.

FIG. 12A shows a representative reaction for epitope detection of 11-meroverlapping peptides (SEQ ID NO:162-165) for spots numbered 214 through217. The highly reactive SPOTs 214 through 217 for positive control seraof women indicates the epitope sequence of 643-VEFKLNYKVTY-653 (SEQ IDNO:163). (FIG. 12B). Likewise, the 216 and 217 peptides reactive withpositive control sera of men suggest the sequence 649-YKVTYS-653 as theepitope. No reactivity was seen with negative control sera of eitherwomen or men. The MAb HA423 epitope is 643-VEFKLNYKVTY-653 (SEQ IDNO:163). FIG. 12C shows the strong dot-blot reaction by positive controlsera of women and men to ACT-P2 immobilized on nitrocellulose. We thensynthesized 15-mer peptides overlapping W10/M2 (AQPLYDEAIAFKEEV) (SEQ IDNO:101) and W13/M5 (FKDTFKYFDKDKSNS) (SEQ ID NO:102), epitopes fromTable 11 (epitopes underlined), and immobilized 1 μg of each peptidetogether and probed with positive control sera. The sera of both womenand men reacted with the combined 15-mer peptide epitopes (FIG. 12D).FIG. 12E presents densitometric scans of the reactive spots and showsthe elevated level of detection by men sera compared to women sera.Finally, FIG. 12F shows the reactivity of individual 15-mer peptidescontaining the epitopes of W9/M1

(SEQ ID NO: 100) (SVNRHHSQLITYIKH),W10/M2, and W13/M5 with positive control sera. FIG. 12G illustrates thedensitometric scans that show men sera giving elevated intensities toW9/M1 and W10/M2 compared to women sera. The extent of detection wasidentical for both sera of women and men to the epitope W13/M5. Negativecontrol sera of both women and men showed no reactions in thesedot-blots of epitopes.

Hydrophobicity and Antigenicity Profiles of the Natural α-ActininSequence.

We then analyzed the immunoreactive epitopes of Table 11 forhydrophobicity and antigenicity. FIG. 13 demonstrates the mapping of theepitopes with the respective profiles and shows that most epitopes werehydrophilic and corresponded with predicted antigenicity. Asrepresentative examples, epitopes W3, W4, W8, HA423/M4/W12, and M5/W13each gave prominent hydrophilic and antigenic characteristics that werenot inconsistent with serum antibody detection.

Sequence Alignment of the T. vaginalis α-Actinin with Proteins of OtherRepresentative Organisms and of the HuACTN1.

BLAST of T. vaginalis α-actinin amino acid sequence (SEQ ID NO:157) ispresented in FIGS. 14A, 14B, 14C, and 14D and shows little amino acidpercent identity with α-actinin-like proteins of different species. Thelow percent identity of amino acids is particularly noteworthy for theboxed epitope sequences indicated above the T. vaginalis amino acidsequence detected by both positive control sera of women and men. Table12 summarizes the percent amino acid sequence identity comparisons forthe individual epitopes compared with sequences for T. suis, a relatedtrichomonad, and the yeasts C. albicans, and S. cerevisiae.

The organisms shown in FIGS. 14A, 14B, 14C, and 14D and Table 12 werechosen because of their relation to STIs and/or eukaryotic pathogens.The range of amino acid percent identity was from 0% to 25%. The percentamino acid identity for α-actinin of T. vaginalis compared with theHuACTN1 was 0% to 50%. Not unexpectedly, the seemingly high percentsequence identity of any epitope with the corresponding region ofHuACTN1 decreased when neighboring amino acids were analyzed. Specificsynthesized peptide epitopes of α-actinin with low to no amino acidsequence identity with other proteins were reactive with positivecontrol sera of both women and men, as shown for representative epitopesin FIG. 12.

Discussion

Research in our laboratory led to the development of the lateral flow,immunochromatographic diagnostic for detection of trichomonad protein infemale patients, and this diagnostic was commercialized and is currentlyin use in the United States and other countries (OSOM® Trichomonas RapidTest, Sekisui Diagnostics, San Diego, Calif.). This diagnostic,developed in our laboratory, works on neither urine nor secretionsobtained from male patients nor on urine spiked with lysates of total T.vaginalis proteins based on our analysis in the laboratory. Reportssuggesting a relation between seropositivity to the ACT-P2 of T.vaginalis and prostate cancer reveal the need for a serum-basedpoint-of-care diagnostic that utilizes a highly specific target. Ourdata show that the 558-amino acid ACT-P2 is a good target for detectionof antibody in both women and men seropositive to T. vaginalis. Thisregion of ACT-P2 was found to possess less homology with otherα-actinin-related proteins, further reinforcing its diagnostic value.The fact that the epitopes detected by the positive control sera of menare located toward the C-terminus revealed why ACT-P2 was a good targetfor our earlier screening for serum antibody. It is noteworthy thatthere is absent or little identity between the peptide-epitope sequenceswith other proteins in databanks and among proteins for T. suis, C.albicans, and S. cerevisiae and the HuACTN1 of humans (Table 12 andFIGS. 14A, 14B, 14C, and 14D). This suggests strongly that highseropositivity is the result of exposure to T. vaginalis. Furthermore,of particular interest is that 15-mer synthetic peptide-epitopes foundwithin ACT-P2 immobilized onto nitrocellulose were detected by sera ofboth positive control sera women and men reactive to α-actinin (FIG.12). Perhaps not surprisingly, most of the peptide epitope amino acidsequences represented portions of the protein that were hydrophilic andantigenic (FIG. 4). The representative 15-mer synthetic peptidescorresponding to W10/M2 and W13/M5 that were readily detected onimmobilized surfaces were in fact highly hydrophilic (FIG. 12). Thisfurther suggests that the reactive 15-mer epitopes represent linear,readily detectable epitopes. A cocktail of or a recombinant proteinencoding for a series of highly-reactive epitopes, such as a rSOE orSOP, represents diagnostic targets.

It is not surprising that α-actinin represents a target forserodiagnostic. This is one of the most immunogenic proteins of T.vaginalis. Its function is to associate with actin, which is importantbecause of the dramatic and rapid morphologic transformation that thisorganism undergoes immediately following contact with vaginal epithelialcells, prostate epithelial cells (unpublished data), and extracellularmatrix proteins, such as laminin and fibronectin. Indeed, recenttranscriptomic and proteomic analyses has revealed the dramaticincreased expression levels of α-actinin required for cytoskeletalrearrangements for morphological changes upon adherence to vaginalepithelial cells and binding to fibronectin. Further, equally elevatedamounts of mRNA encoding for trichomonad GAPDH and α-enolase were found,and both of these proteins are surface ligands for binding fibronectin.There are four α-actinin human homologs, none of which are crossreactivewith the MAb HA423 to the trichomonad α-actinin (FIG. 9 and Table 15)and with positive control sera of women and men. These human α-actininproteins are known to have a less conserved central region, as is thecase for all actin-binding proteins of the spectrin family.

Equally noteworthy is that the epitopes detected by MAb HA423 andpositive control sera of women and men are invariant. Laboratory-adaptedT. vaginalis isolates grown in batch culture for >20 years possess theMAb 423-immunoreactive α-actinin with the same M_(r). Further, more thanfifty fresh clinical isolates, one-half of which are the Type II P270phenotypically-varying isolates with the dsRNA virus, all possessα-actinin detected by MAb and positive control sera of women and men. Wehave seen no relation between T. vaginalis with or without Mycoplasmaand changes with α-actinin. Thus, this invariant and stable immunogenicprotein appears suitable for a rapid serodiagnostic test fortrichomonosis.

Of interest is the number of epitopes detected by the positive controlsera of women patients compared to sera of men. This may be the resultof different presentation of the protein(s) during immune surveillancethat results from the unique urogenital regions of women in contrast tomen. It is known to those of ordinary skill in the art that womenpatients with trichomonosis possess IgG antibody in the serum and vaginato numerous trichomonad proteins, perhaps indicating a more vigorousantibody response during infection compared to men. Studies by othersdemonstrated the highly immunogenic nature of and serum antibodyresponse by women to α-actinin. Nonetheless, these data show that menrespond to exposure to T. vaginalis by producing serum IgG antibody,especially to the epitopes located toward the carboxy-terminal regionwith the least identity to other known proteins. Importantly, whatremains unknown is the temporal relationship between seropositivity withinitial exposure to this STI, and this critical absence of clinicalinformation perhaps may be corrected through future availability of aserodiagnostic for women and men. What is known, however, albeit in onlya small sample size is that one week after treatment of women withtrichomonosis the vaginal antibody to proteinases was not detected.

The literature is replete with examples of peptide epitopes utilized fordiagnostics of infectious diseases. For example, rapid diagnostics forPlasmodium falciparum employ epitopes of histidine-rich proteins.Diagnosis of visceral leishmaniasis is performed with rapidantigen-based tests, and specific epitopes of the proteins p120 and p140are used for detection of Ehrlichia chaffeensis and E. canis,respectively. This shows the value of characterization of immunogenicepitopes for developing specific targets for serodiagnosis.

In summary, our results present evidence for the validity of α-actininand the truncated ACT-P2 as a target for serodiagnosis in both women andmen exposed to T. vaginalis. This is important not only for screeningmen possibly exposed to this STI in relation to the possibility ofprostate cancer development but for a more rapid, non-invasive test forwomen as well. This approach highlights the methods by which peptideepitopes of immunogens may be identified as targets for antibodydetection for determining exposure to and infection by this significantSTD pathogen.

Example 10. α-Actinin String-of-Pearls (KK-ACT-SOP) Epitopes, and SOESequences

Sequences are shown in Table 15, comprising 229 amino acids; pI=9.93;MW=27,207 Da.

TABLE 15 T. vaginalis α-Actinin epitopes, and SOE sequences EPITOPEEPITOPE # NAME AA SEQUENCE SEQ ID NO: 107  1 W1 SV RREGLLDDAWE KTSEQ ID NO: 108  7 W2 LARQ IQFETIET DFE SEQ ID NO: 109  3 W3 PSKWH KQPKMMVQKR SEQ ID NO: 110  4 W4 QG YEHVAVNNFTT SW SEQ ID NO: 111  5 W5 GIYVYLDPEDVID TT SEQ ID NO: 112  6 W6 KIAAM ADKIK RTVAI SEQ ID NO: 113  7W7 IPGI RGKLASVI SYN SEQ ID NO: 114  8 W8 CKSG NRPIPEIP QGLSEQ ID NO: 100  9 W9/M1 SVNR HHSQLITY IKH SEQ ID NO: 101 10 W10/M2 AQPLYDEAIAFK EEV SEQ ID NO: 117 11 W11/M3 ELVEF KLNYK VTYTY SEQ ID NO: 11817 W12/M4/HA423 EFKLN YKVTY TYSDA SEQ ID NO: 102 13 W13/M5 FKDTF KYFDKDKSNS SEQ ID NO: 120 KK-SV RREGLLDDAWE KT-KK-LARQ IQFETIET DFE- KK-PSKWHKQPKM MVQKR-KK-QG YEHVAVNNFTT SW- KK-GI YVYLDPEDVID TT-KK-KIAAM ADKIKRTVAI- KK-IPGI RGKLASVI SYN-KK-CKSG NRPIPEIP QGL- KK-SVNR HHSQLITYIKH-KK-AQPL YDEAIAFK EEV- KK-ELVEF KLNYK VTYTY-KK-EFKLN YKVTY TYSDA-KK-FKDTF KYFDK DKSNS-KK-HHHHHH SEQ ID NO: 120 KKSV RREGLLDDAWE KTKKLARQIQFETIET DFEKKPS KWH KQPKM MVQKRKKQG YEHVAVNNFTT SWKKGI YVYL DPEDVIDTTKKKIAAM ADKIK RTVAIKKIPGI RGKLAS VI SYNKKCKSG NRPIPEIP QGLKKSVNRHHSQLITY IK HKKAQPL YDEAIAFK EEVKKELVEF KLNYK VTYTYKKE FKLN YKVTYTYSDAKKEKDTF KYFDK DKSNSKKHHHHH H SEQ ID NO: 12KKSVRREGLLDDAWEKTKKLARQIQFETIETDFEKKPSKWHKQPKMMVQKRKKQGYEHVAVNNFTTSWKKGIYVYLDPEDVIDTTKKKIAAMADKIKRTVAIKKIPGIRGKLASVISYNKKCKSGNRPIPEIPQGLKKSVNRHHSQLITYIKHKKAQPLYDEAIAFKEEVKKELVEFKLNYKVTYTYKKEFKLNYKVTYTYSDAKKFKDTFKYFDKDKSNSKKHHHHH H

Example 11. Examples of T. pallidum Highly Immunogenic Peptides, 15-MerEpitopes, and SOE as Targets for Serodetection, Shown in Table 16

Epitope sequences identified as SEQ ID NO: 121-126 are highlyimmunogenic (see Brinkman, Antoni, and Liu cited below). Additionalimmunogenic protein sequences for T. pallidum are provided in SEQ ID NO:161-164. Based on this information, and using the methods of thedisclosure, the SOE of SEQ ID NO: 127 was designed and synthesized.Experimental evidence shows that it is able to detect T. pallidum in abiological sample, and to elicit an immune response when injected into asubject. The H₆ (also referred to as hexa-His) at the amino terminal endof the SOE polypeptide is for purification of the SOE using Ni-NTA(nickel) affinity chromatography.

TABLE 16 T. pallidum epitopes and SOE sequences AA SEQUENCESEQ ID NO: 121 LSTSLLTTCDFTGIFA SEQ ID NO: 122 IQSEVPIK SEQ ID NO: 123LLIGGSRGYGEIKLE SEQ ID NO: 124 RPDLYAAVGE SEQ ID NO: 125ASGAKEEAEKKAAEQRALL SEQ ID NO: 126 EVEDVPKVVEPASEREGGER SEQ ID NO: 127KK-LSTSLLTTCDFTGIFA- KK-IQSEVPIK- KK-LLIGGSRGYGEIKLE- KK-RPDLYAAVGE-KK-ASGAKEEAEKKAAEQRALL- KK-EVEDVPKVVEPASEREGGER- KK-HHHHHH

-   Brinkman, M B, et al., 2008. A novel Treponema pallidum antigen,    TP0136 . . . Infect. Immun. 76:1848-1857.-   Antoni, G., et al., 1996. Detection of antigen determinants in the    Treponema pallidum . . . 189:137-140.-   Liu, H., et al., 2007. Molecular characterization and analysis of a    gene . . . 56:715-721.

Example 12. Examples of N. gonorrhoeae Highly Immunogenic Peptides,15-Mer Epitopes, and SOE as Targets for Serodetection, Shown in Table 17

Epitope sequences, SEQ ID NO: 128-133, were derived from Cooke et al.,1997; 143:1415-1422. Additional immunogenic protein sequence for N.gonorrhoeae is provided in SEQ ID NO: 165. Based on this information,SOE with sequence provided in SEQ ID NO: 134 was designed andsynthesized. Experimental evidence shows that it is able to detect N.gonorrhoeae in a biological sample, and to elicit an immune responsewhen injected into a subject. The H₆ at the amino terminal end of theSOE polypeptide sequence is for purification of the SOE using Ni-NTA(nickel) affinity chromatography.

TABLE 17 N. gonorrhoeae highly immunogenicpeptides encoded in 15-mer epitopes, and SOE. AA SEQUENCE SEQ ID NO: 128FGSKIGFKGQEDLGN SEQ ID NO: 129 GFSGSVQYAPKDNSG SEQ ID NO: 130GFFAQYAGLFQRYGE SEQ ID NO: 131 VEKLQVHRLVGGYDN SEQ ID NO: 132NSHNSQTEVAATAAY SEQ ID NO: 133 NTYDQVVVGAEYDFS SEQ ID NO: 134KK-FGSKIGFKGQEDLGN- KK-GFSGSVQYAPKDNSG- KK-GFFAQYAGLFQRYGE-KK-VEKLQVHRLVGGYDN- KK-NSHNSQTEVAATAAY- KK-NTYDQVVVGAEYDFS- KK-HHHHHH

Example 13. Production of a SOE Chimeric Construct Using Epitopes of T.vaginalis Fructose-1,6-Biphosphate Aldolase, α-Enolase, and GAPDHwithout Sequence Identity to Bacterial, Fungal, Parasite, and HumanProteins

TABLE 18 List of synthetic peptides ofrepresentative T. vaginalis epitopes of ENO,GAP, and ALD Mabs for reactivity withimmunoreactive women and men sera. ALD13 andALD32 refer to MAbs. Immunogenic residuesdetected by women and men sera are underlined. women/men epitopeamino acid designation sequence SEQ ID NO: 166 E-ALD13 AEHDAIVKECIAEAASEQ ID NO: 167 E-W3, E-M3 TDGTVLKKNIGGNAS SEQ ID NO: 168 E-M7ILVKKYGLSAKNLDEF SEQ ID NO: 169 E-M8 ASSEFYDEEKKLYEV SEQ ID NO: 170 E-M9DYENWTKLNARLGQRV SEQ ID NO: 171 G-M1 RASRKLYPKDIQVVA SEQ ID NO: 172G-W1, G-ALD32 NVYLLKYDTAHRAFP SEQ ID NO: 173 G-W3 VVLESTGIFRTKAEKSEQ ID NO: 174 G-W6 TLNNAFGIRNGFMTTV

The epitopes in Table 18 were combined with 9 additional epitopes asdescribed herein to produce a SOE (“AEG:SOE2) having 18 epitopes linkedwith glutamic acid repeats (FIG. 15; SEQ ID NO: 175). The SOE has anisoelectric point of about 5.05 and a molecular weight of about 35.9 kDa(FIGS. 15-16). The SOE is reactive with pooled positive women/men seraand mouse anti-Tv serum but not with the pooled negative controlwomen/men sera (FIG. 17).

The activity of the SOE was then compared to T. vaginalis α-actinin, animmunogenic trichomonad protein not found among other microorganisms(FIG. 19).

When compared to α-actinin, the SOE had a very high sensitivity,specificity, and predictive value when used as a serum diagnostic of T.vaginalis (FIG. 18).

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

The invention claimed is:
 1. A method for detecting the presence of oneor more Trichomonas vaginalis microorganisms in a biological sample of asubject, comprising the steps of: combining said biological sample witha polypeptide including a series of epitopes (SOE) which includes atleast a plurality of epitopes selected from the group consisting of SEQID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO:170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174,said epitopes being arranged as a linear array with each of saidepitopes being connected by an amino acid linker, wherein said combiningis performed under conditions whereby antigen-antibody complexes arepermitted to form; and detecting formation of at least oneantigen-antibody complex as an indication of a presence of at least onemicroorganism of said one or more Trichomonas vaginalis microorganismsin said biological sample.
 2. The method of claim 1, wherein the SOEincludes at least six epitopes.
 3. The method of claim 1, wherein saidTrichomonas vaginalis microorganisms are selected from the groupconsisting of T. vaginalis isolates T016, T068-II, UT40, and VB102. 4.The method of claim 1, wherein said detecting step is performed using animmunoassay.
 5. The method of claim 4, wherein said immunoassay is anenzyme-linked immunosorbent assay (ELISA).
 6. The method of claim 1,wherein said biological sample is selected from the group consisting ofserum, plasma, blood, saliva, semen, cerebrospinal fluid, semen,prostatic fluid, urine, sputum, joint fluid, body cavity fluid, wholecells, cell extracts, tissue, biopsy material, aspirates, exudates,vaginal washings, pap smear samples, pap smear preparations, slidepreparations, fixed cells, and tissue sections.
 7. The method of claim1, wherein said subject is selected from the group consisting of human,non-human primate, dog, cat, cattle, sheep, swine, horse, bird, mouse,and rat.
 8. The method of claim 1, wherein the SOE has the sequence ofSEQ ID NO:
 175. 9. The method of claim 1, wherein the isoelectric pointof the SOE is in a range from 4.90 to 5.10.